Oxidative stress & hypoxia Pey-Jium Chang

Oxidative stress & hypoxia








Pey-Jium Chang



















Oxidative Stress




Free radicals
-- any molecular species with one or more unpaired electron(s), a characteristic that provides them with an “unstable” chemical
nature and thus confers them with the property
of being very reactive.



Types of free radicals:
Reactive oxygen species (ROS): superoxide
(O2-), hydroxyl (OH-), hydroperoxyl (HOO-), peroxyl (ROO-) and alkoxyl (RO-) radicals



Reactive nitrogen species (RNS)











Various ROS-producing enzyme systems



Mitochondrial electron transport chain
Cytochrome P450
Lipoxygenase
Cyclooxygenase
NADPH oxidase complex
Xanthine oxidase
Peroxisomes


An imbalance between free radical-generating and radical- scavenging systems results in oxidative stress.



A chronic ROS exposure is carcinogenic, while
excess levels of ROS are toxic to cancer cells.







































(Clin Cancer Res, 2007, 13: 789-794)













Impact of free
radicals released at sites of inflammation on cellular molecules






















(Nat Rev Cancer, 2003, 3:276-285)

Examples of oxyradical overload diseases












Free-radical
generation, cellular stress and tumorigenesis























(Nat Rev Cancer, 2003, 3:276-285)




Association between human cancers and gene
polymorphisms in DNA repair and antioxidation


Chemoprevention in oxyradical overload diseases

Chronic inflammation and production of free radicals regulate multiple cellular processes.












































(Nat Rev Cancer, 2003, 3:276-285)






ROS-induced oxidative stress based epigenetic
alterations in human carcinogenesis
























(Gastroenterology, 2008, 135:2128-2140)

ROS down-regulate E-cadherin expression in HCC cell lines

Hep3B cells Immunoblot
(NAC: antioxidant)

Real-time RT-PCR


































immunofluorescence



Menadione treatment



PMS
treatment






MMP activity and expression increases in
H2O2-treated cells





Gelatin zymography Real-time RT-PCR Matrigel invasion assay

ROS up-regulate Snail expression in HCC cell lines




H2O2 treatment menadione treatment PMS treatment


Snail knockdown





















Real-time RT-PCR








H2O2 Snail E-cadherin



Involvement of PI3K/Akt/GSK3 pathway in Snail
regulation by H2O2




Phospho-MAPK assay

LY294002: PI3K inhibitor



Real-time RT-PCR






























H2O2

PI3K Akt GSK3

Snail E-cadherin

ROS induce methylation of the E-cadherin promoter



Methylation-specific PCR






ROS induce the recruitment of HDAC1 and DNMT1 in
the E-cadherin promoter







Chromatin immunoprecipitation Chromatin immunoprecipitation

Snail overexpression induces methylation of the E-cadherin promoter and binding of HDAC1 and DNMT1





Time course

shRNA knockdown






















ChIP ChIP

Methylation of the E-cadherin promoter


ChIP-MSP- sequencing ChIP-MSP- sequencing






















ChIP-Bisulfite sequencing




Snail interacts with HDAC1 and DNMT1






Reporter assay






























ChIP



Co-immunoprecipitation


Snail up-regulaton and promoter methylation are correlated with down-regulation of E-cadherin in HCC


Snail up-regulaton and promoter methylation are correlated with down-regulation of E-cadherin in HCC



Molecular mechanism of ROS-induced
E-cadherin down-regulation




















Hypoxia



Hypoxia on tumor biology





Hypoxia












Receptor tyrosine kinase-mediated signaling



Angiogenesis


Vasculogenesis


EMT

Loss of genomic
stability



Suppression of immune reactivity



Metastasis



Invasiveness

Mechanisms of resistance of hypoxic cells to therapy




Hypoxia and clinical outcomes in human cancer

Hypoxia-responsive signaling pathway















































(Nat Rev Cancer, 2011, 11: 393-410)




Control of hypoxia-inducible factor by hydroxylation
































(Nat Rev Cancer, 2008, 8: 865-873)


FIH1 (factor inhibiting HIF-1): Asparaginyl hydroxylase
PHD2: prolyl hydroxylase domain-containing protein
VHL: von Hippel-Lindau

Hypoxic mitochondria signal via ROS to stabilize HIF-1
and activate MAP kinase signaling











































(Clin Cancer Res, 2007, 13: 789-794)

Hypoxia-responsive signaling pathway


DNA double-strand breaks in irradiated hypoxic cells










































(Nat Rev Cancer, 2008, 8: 180-192)


Model of hypoxia-mediated genetic instability












































(Nat Rev Cancer, 2008, 8: 180-192)

Hypoxia-responsive signaling pathway





































(J Clin Invest 2010, 120: 127-141)


Molecular mechanism of autophagy

Autophagy is primarily present in hypoxic tumor regions





Human head and neck xenografts






































Pimonidazole (pimo): binding to SH-containing molecules in hypoxic tissues






Autophagy is induced by hypoxia




Regulation of autophagy genes LC3B and ATG5 during
hypoxic exposure




Induction of LC3B and ATG5 is dependent on PERK signaling



Knockdown of the indicated gene in cells






























PERK ATF4 CHOP

LC3B ATG5


















ATF4 ChIP analysis
(LC3B promoter)



CHOP ChIP analysis
(ATG5 promoter)




Inhibition of autophagy by chloroquine, a
lysosomotrophic drug, sensitizes cells to hypoxia

Model for recycling and regeneration of LC3B during hypoxia

Molecular targets in hypoxic cells

Oxygen dependence of hypoxia-responsive processes in tumors







Necrosis

Pimonidazole
CA9 (HIF-1 target)





EF5






HIF-1




radiation










UPR

















Blood vessels

Mechanisms of metabolic activation of bioreductive prodrugs

Structures of bioreductive prodrugs

Bioreductive prodrugs in clinical development