CANCER CENTER KAROLINSKA
KAROLINSKA INSTITUTET
Oxidative stress and cancer therapeutics
Stig Linder
DEP OF MEDICINE AND HEALTH LINKÖPING UNIVERSITY
Principle of the mechanism(s) of action of most cancer therapeutics: To explore the difference between proliferating and non-proliferating cells to DNA damage and mitosis inhibition.
Oxidative stress has been implicated in the side effects of of several cancer drugs Examples: • Ototoxicity of cisplatin • Cardiac toxicity of doxorubicin
Many cancer therapeutic drugs induce oxidative stress
How strong is the evidence for oxidative stress being instrumental to therapeutic effects?
Many cancer therapeutic drugs induce oxidative stress
How strong is the evidence for oxidative stress being instrumental to therapeutic effects?
For many drugs quite strong, for others not
A common source of problems in this field is the use of N-acetyl cysteine (NAC) Common structure of paper: (1) Compound X induces cell death and apoptosis of cancer
cells (Figure 1, 2)
(2) Compound X induces mitochondrial depolarizaton, caspase-9, caspase-3 and PARP cleavage (Figure 3)
(3) Compound X induces p53 + other favorite markers (Figure 4)
(4) Compound X induces ROS (DCFDA) (Figure 5)
(5) The effect of compound X is abrogated by NAC (Figure 6) Conclusion: drug X induces apoptosis by the intrinsic pathway and apoptosis is dependent on oxidative stress.
Cys-SPiperlongumine contain two Michael acceptors that are expected to bind NAC (or GSH) - Is the effect of NAC not scavenging of ROS but inactivation of the drug (piperlongumine)?
Cisplatin – a standard chemotherapeutic agent reported to induce oxidative stress
ROS implicated both in the therapeutic effect on cancer cells and in side effects (ototoxicity) Many studies use NAC
• Use of NAC • Use of very high concentrations of cisplatin
Drugs – molecular biology
Drugs - pharmacology
Important to use drugs at relevant concentrations (IC50 – IC90)
Is 0.3 mM cisplatin a relevant concentration?
How to find out?
Therapeutic window of cisplatin and radiation: The preferential sensitivity of proliferating cells to DNA damage
Drug/radiation
DNA damage/mitotic catastrophe
Senescence
Apoptosis
DNA damage/mitotic catastrophe
Day 1 Day 2 Day 3 Day 4
At the IC50 concentration, cisplatin induces DNA damage-induced senescence, mitotic catastrophe and secondary apoptosis. Apoptosis is secondary to irreversible cellular damage: Apoptosis: a ”funeral mechanism” – not a ”death mechanism” Studying the mechanism(s) of apoptosis is unlikely to be relevant for the understanding of cisplatin sensitivity/resistance of tumors. Studies of funerals is of limited interests for the understanding of life, death and disease.
Cisplatin and radiation and molecular biology Unphysiological doses of drug/radiation
Drug/radiation (10-100x dose)
DNA damage/mitotic catastrophe
Senescence
Apoptosis
Day 1 Day 2 Day 3 Day 4
Studies of signaling events
Acute apoptosis
Acute apoptosis (over 24 hours) is induced at concentrations of 10 – 30 µM We showed > 10 years ago that ”acute” apoptosis is an off-target effect: Mandic et al., Mol Cell Biol 2002, Mandic et al., J Biol Chem 2003 Berndtsson et al., Int J Cancer 2007 Oxidative stress, calcium release from the ER, activation of calpain, apoptosis
Cisplatin (20 µM) induces apoptosis in enucleated cells (=off-target), a process inhibited by NAC (as expected) and also by the Vitamin E analogue Tiron
Cisplatin induces senescence (prolonged growth arrest) at lower doses, a process not abrogated by the antioxidant Tiron
Cisplatin and radiation and molecular biology Unphysiological doses of drug/radiation
Drug/radiation (10-100x dose)
DNA damage/mitotic catastrophe
Senescence
Apoptosis
Day 1 Day 2 Day 3 Day 4
ER stress, calcium release, oxidative stress
Acute apoptosis
PubMed: 1566 hits on apoptosis + mechanism + cisplatin 2001-2005 245 2006-2010 411 2010-2015 774 2016 175 (x5 = 1286 studies 2016-2020?)
PubMed: 7744 hits on apoptosis + cisplatin Cost of producing a scientific paper? Europe/USA/Asia 500,000 Skr? Cost of studying a biologically irrelevant off-target effect: 1 billion SKr?
The same problem for other anticancer drugs 5-FU: IC50 in the NCI60 cell line panel: 6 µM Use in apoptosis research: 800 µM (!)
Summary: Cancer therapeutics is a very important research area • Important to use drugs at relevant concentrations Oxidative stress has been implicated in the mechanisms of action of several cancer drugs • Important to use different scavengers, to make sure that the drug is not inactivated by the scavenger
Oxidative stress has been implicated in the mechanisms of action of several cancer drugs Example from our own research: proteasome inhibitors
Degradation of substrate proteins requires the removal of ubiquitin units by cysteine deubiquitinases (DUBs) USP14 and UCHL5
and by the DUB POH1
USP14
UCHL5
USP14
UCHL5
POH1
The human genome encodes ~460 active proteases The human genome encodes ~80 active deubiquitinases (DUBs; ubiquitin isopeptidases): Ubiquitin-specific proteases (USPs) Cysteine protease Ubiquitin C-terminal hydrolases (UCHs) Cysteine protease Ovarian tumor (OTs) Cysteine protease Josephins (Machado–Joseph disease) Cysteine protease JAMM/MPN+ metalloenzymes Zinc metalloproteases Vast majority of DUBs are cysteine proteases – expected to be highly druggable
b-AP15 and VLX1570 block the proteasome deubiquitinases (DUBs) USP14 and UCHL5 – resulting in inhibition of protein
degradation
USP14
UCHL5
b-AP15 VLX1570
D’Arcy et al., Nature Medicine 2011; Tian ZZ et al., Blood (2013); Wang et al., Molecular Pharmacology 2014; Wang et al., Chem Biol Drug Design 2015; Wang et al., Scientific Reports (2016).
b-AP15/VLX1570 represent a novel class of inhibitors of proteasome function Distinct mechanism is expected to result in therapeutic effects on cells resistant to bortezomib/carfilzomib
b-AP15 VLX1570
Bortezomib Carfilzomib
USP14 UCHL5
Preferential inhibition of USP14 (band = active enzyme)
19 S proteasomes in vitro, Ub-VS labeling
VLX1570
VLX1570 binds to proteasomal DUBs in vitro and in myeloma cells
(Wang et al., Scientific Reports 2016)
Cellular thermal shift assay (stabilization of protein after drug binding)
b-AP15/VLX1570 are effective in SCID mouse models of multiple myeloma
KMS11-LUC multiple myeloma cells in SCID mice were treated with vehicle or b-AP15/VLX1500 (4 mg/kg/I.P.).
20 40 60
100
Days
Sur
viva
l
50 Vehicle
b-AP15
Tian ZZ, D'Arcy P, Wang X, Ray A, Tai Y-Z, Hu Y, Carrasco RD, Richardson P, Linder S, Chauhan D and Anderson K. (2013) Blood, EPub Dec 6, 2013.
Is proteasome inhibition the mechanism underlying cell death?
Drug
Target A Target B Target C Target D Target E
Apoptosis Apoptosis
Monitoring accumulation of a Ub-YFP reporter
Unstable Ub-YFP fusion protein accumulates in cells with impaired proteasome function
X
Strict correlation between proteasome blocking and cell death
X
Genetic approaches
Knock-down of USP14/UCHL5 phenocopies the effect of b-AP15 (proteasome blocking and induction of apoptosis)
Wang et al., Molecular Pharmacology 85 (2014) 932
Gene expression analysis (CMAP)
Expose cells to drug for 6 hours -> microarray -> compare pattern of induced gene expression profile with dase base (CMAP)
Molecular mechanism underlying phenotypic effect
• Dose response studies –phenotypic effect at the same drug concentration as target inhibition? • Genetic approaches (knock-down, CRISPR, mutations) • Pharmacological approaches (inhibition of down stream targets etc), studies of signaling • Gene expression analyses
Initial observation: strong induction of the Nrf2 target gene hemeoxygenase-1 (HO-1, HMOX1)
b-AP15 induces oxidative stress
b-AP15 1 hr, wash, then add NAC
Induction of ROS, scavengers inhibit apoptosis
Proteotoxic stress ER stress
Proteasome inhibition
Oxidative stress
Apoptosis
b-AP15 inhibits thioredoxin reductase
Proteotoxic stress ER stress
Proteasome inhibition
Oxidative stress
Apoptosis
TrxR inhibition
Proteotoxic stress ER stress
Proteasome inhibition
Oxidative stress
Apoptosis
TrxR inhibition
Proteotoxic stress ER stress
Proteasome inhibition Oxidative stress
Apoptosis
TrxR inhibition
Oxidative stress response is weaker in Rho0 cells
Mitochondrion
Disturbed electron transport
ROS
Apoptosis
Soluble misfolded proteins with exposed hydrophobic sequences (not contained in aggresome)
b-AP15 inhibits thioredoxin reductase
Proteotoxic stress ER stress
Proteasome inhibition
Oxidative stress
Apoptosis
TrxR inhibition
Proteotoxic stress ER stress
Proteasome inhibition
Oxidative stress
Apoptosis
TrxR inhibition
Proteotoxic stress ER stress
Proteasome inhibition Oxidative stress
Apoptosis
TrxR inhibition
b-AP15 inhibits thioredoxin reductase
Proteotoxic stress ER stress
Proteasome inhibition
Oxidative stress
Apoptosis
TrxR inhibition
Proteotoxic stress ER stress
Proteasome inhibition
Oxidative stress
Apoptosis
TrxR inhibition
Proteotoxic stress ER stress
Proteasome inhibition Oxidative stress
Apoptosis
TrxR inhibition
Oxidative stress during cancer therapy:
Therapeutic effects
Side effects
Artifacts
Oxidative stress during cancer therapy:
Complex question, needs to be carefully addressed using appropriate controls, careful choice of reagents and adequate drug concentrations.
ACKNOWLEDGEMENTS
LINKÖPING UNIVERSITY PADRAIG D’ARCY XIN WANG KARTHIK SELVARAJU NAN ZHANG CANCERCENTER KAROLINSKA MARIA HÄGG OLOFSSON SLAVICA BRNJIC ELLIN HILLERT MAGDA MAZURKIEWICZ DANA FARBER CANCER INSTITUTE DHARMINDER CHAUHAN, KEN ANDERSON MAYO CLINIC (JAX) ASHER CHANAN-KHAN ISTITUTO TUMORI, MILAN PAOLA PEREGO VIVOLUX AB HANS ROSÉN, PER HAGMAR