2017 redox course - university of...

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.

Many cancer therapeutic drugs induce oxidative stress

Therapeutic effects

Side effects

Artifacts

Oxidative stress has been implicated in the side effects of of several cancer drugs Examples: • Ototoxicity of cisplatin • Cardiac toxicity of doxorubicin


How strong is the evidence for oxidative stress being instrumental to therapeutic effects?


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.

PL = piperlongumine

Normal cells

Tumor cells

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)?

Piperlongumine is in fact a proteasome inhibitor

NAC scavenges ROS but also inhibits both the proteasome inhibitory activity 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?

Over 94 cancer cell lines, IC50 for cisplatin is ~ 1 µM

Over 94 cancer cell lines, IC50 for cisplatin is ~ 1 µM Why use a drug at 100 – 300 fold its IC50?

Fractionated radiotherapy: 1 Gy per day

Therapeutic window of cisplatin and radiation: The preferential sensitivity of proliferating cells to DNA damage

Drug/radiation

DNA damage/mitotic catastrophe

Senescence

Apoptosis


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)


Senescence

Apoptosis


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

20 µM cisplatin 20 µM cisplatin

ROS

Apoptosis

DNA damage correlates to inhibition of clonogenic outgrowth but not to (acute) 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)


Senescence

Apoptosis


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

The ubiquitin-proteasome system

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

Mechanism of oxidative stress induction?




Oxidative stress

Apoptosis

TrxR inhibition



Oxidative stress

Apoptosis

TrxR inhibition


Proteasome inhibition Oxidative stress

Apoptosis

TrxR inhibition

Auranofin, a thioredoxin reductase inhibitor, does not inhibit the proteasome

Misfolded proteins interact with mitochondria – another source of ROS?

Mitochondrial oxygen consumption and morphology altered – but mitophagy is not induced

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)




Oxidative stress

Apoptosis

TrxR inhibition



Oxidative stress

Apoptosis

TrxR inhibition


Proteasome inhibition Oxidative stress

Apoptosis

TrxR inhibition

Model of proteotoxicity in relation to mitophagy

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

2017 redox course - university of...

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