# Presentation: 051096

## Redox Biology: Introduction

**Terry W. Moody, Ph.D.**
**NCI Center for Cancer Research**
**Office of Training and Education**

## Organizing Committee

**Terry Moody**
**Jonathan Wiest**
**David Wink**

## NCI CCR OTE Courses

**●  ****Translational Research in Clinical Oncology (TRACO)**
**●  ****Statistical Analysis of Research Data**
**●  ****Redox Biology**
**●  ****Mouse Research Models**
**●  ****Cancer Biotechnology**
**●  ****Cancer Scientific Writing**
**●  ****Writing Cancer Grant Applications**
**●  ****Scientific Management Training**
**●  ****Teaching in Medical Education**

## Redox Biology lecture schedule(Bldg. 50, Rms. 1227/1233, 4-6 p.m.)

**Date****	****	****Lecturer****	****	****TOPIC**
**Sept. 30****	****Moody****	****	****Introduction**
** ****	****	****	****Wink****	****	****Chemistry**
**Oct. 7****	****REDOX WORKSHOP**
**Oct. 14****	****Wink****	****	****Biology**
**	****	****	****Moody****	****Signal Transduction**
**Oct. 21****	****Roberts****	****	****Angiogenesis**
**	****	****	****Pluta****	****	****CNS**

## Redox Biology

**Date****	****	****Lecturer****	****	****Topic**
**Oct. 28****	****Ambs****	****	****	****Epidemiology**
**	****	****	****Hussain****	****	****Inflammation**
**Nov. 4****	****Yeh****	****	****	****Carcinogenesis**
**	****	****	****Young****	****	****Immunology**
**Nov. 11****	****NO CLASS**
**Nov. 18****	****Leto****	****	****	****NADPH Oxidases****	****	****	****Krishna****	****	****Imaging**
**Nov. 25****	****Redox enzymes****	****Ridnour****	****	****	****	****Mitchell****	****	****Cancer therapy**
**	****	****	**

## October 7, 2008 NCI CRBF Workshop

**Time****	****	****Lecturer****	****	****Topic**
**10:30 a.m.****	****Rigas****	****	****	****Cancer Prevention**
**11:00 a.m.****	****Mitchell****	****	****Nitroxides****	****	**
**11:30 a.m.****	****Mikkelsen****	****	****Ionizing Radiation**
**12 noon****	****Krishna****	****	****Imaging****	**
**12:30 p.m.****	****Glynn****	****	****	****Epidemiology**
**LUNCH BREAK****	**
**2:30 p.m.****	****Keefer****	****	****	****JSK analogues****	**
**3:00 p.m.****	****Miranda****	****	****HNO**
**3:30 p.m. ****	****Figg****	****	****	****Thalidomides and leukemia**
**4:00 p.m. ****	****Santus****	****	****	****Hydrogen sulfide**
**4:30 p.m. ****	****Switzer****	****	****	****Novel NSAIDs****	****	****	**

## Videocast

**●****The course is open to all interested NIH personnel without charge.**
**●****The course is videoconferenced to NCI-Frederick, Bldg. 549, Rm. A.**
**●****The course is videocast live and is archived as a past event.**** **

## Course certificate

**●****R****egistrants can obtain a course certificate upon passing a computer graded final examination **

## Redox Species

******Reactive oxygen species (ROS)**
**●****Reactive nitrogen species (RNS)**
******Hydrogen sulfide**
******Carbon monoxide**

## Reactive Oxygen Species (ROS)

**●****O****2****- ****(superoxide)**
******OH****● ****(hydroxy radical)**
******H****2****O****2 ****(hydrogen peroxide)**
******O****3 ****(ozone)**
******1****O****2 ****(singlet oxygen)**
******LOOH (lipid peroxides)**
******LOO******** (lipid peroxyl radical)**
******LO******** (lipid alkosyl radical)**

## Cytotoxicity of ROS:

**O****2****.-****	****	****	****H****2****O****2****	****	****	****HO****.**
**(Superoxide)****	****(Hydrogen peroxide)****	****Fe/Cu****	****(hydroxylradical)**
**●****ROS oxidize DNA, lipids and proteins leading to cytotoxicity.**

## ROS can be metabolized by enzymes:

**●****Superoxide dismutase**** protects against O****2****-**
**2 superoxide + 2 H****+ ****> O****2**** + H****2****O****2**
**SOD1 is a homodimer in the cytosol**
**Copper and Zinc are cofactors**
**SOD1 contains 153 amino acids and has a molecular weight of 15,805 Daltons**

## ROS can be metabolized by enzymes:

******Catalase**** protects against H****2****O****2**
**2**** ****H****2****O****2 ****>**** ****O****2 ****+ 2H****2****O**
**Catalase is a homotetramer which is localized to the peroxisome**
**Cofactors include a Heme group and NADP**
**Catalase has 526 amino acids and a molecular weight of 59,625 Daltons**

## ROS can be metabolized by enzymes:

****** ****Glutathione peroxidase**** protects against H****2****O****2**
**2 GSH + H****2****O****2**** > GSSG + 2H****2****0**
**GPX1 is a homotetramer in the cytosol**
**GPX1 has Se attached to Cys****47**
**GPX1 has 201 amino acids and weighs 21,899 Daltons**

## ROS can be metabolized by enzymes:

**●****Peroxiredoxins**** reduce H****2****O****2****, organic, fatty acid and phospholipid hydroperoxides**
**2R’SH + ROOH > R’SSR’ + H****2****O +ROH**
**PRDX6 is a homotetramer present in many organelles**
**Cys****46**** is oxidized to Cys-SOH**
**Cys-SOH may react with CysSH from another subunit to form a disulfide**
**PRDX6 contains 223 amino acids and weighs 24,904 Daltons**

## Oxidants affect two distinct signaling pathways:

**●****MAP kinase/AP-1 (Activator Protein-1) affecting cell proliferation, survival and apoptosis/death.**
******NF-********B (Nuclear Factor-********B) affecting inflammation, survival and cell cycle control.**

## ROS are involved in:

**●****Heart disease**
******Stroke**
******Aging**
******Arthritis**
******Neurodegeneration**
******Cancer**
**●****and the list keeps growing**

## What are the effect of vitamins on cancer? 

**●****Many epidemiological studies show**
**that eating fruit and vegetables prevent**
**heart disease and cancer.**
**● ****Studies on specific vitamins** **and substances have been less promising.**
**●****Studies have focussed on ****Vitamin C,**
**Vitamin E and Vitamin A derivatives**

## Vitamins and Cancer

**●****The research was carried out in France, led by Dr. Serge Hercberg from the Scientific and Technical Institute for Nutrition and Diet in Paris, and was called the SuViMax study. A total of 13,017 men and women aged between 35  and 60 years old were recruited to take part. Half were given a daily supplement containing a combination of antioxidant vitamins, while the others received a placebo pill**
**● ****Patients received ****6 mg beta-carotene, 120 mg of vitamin C, 30 mg of vitamin E, 100 micrograms of selenium and 20 mg of zinc. **
**●****According to the researchers, the pill's lack of effect on women's health risks might be due to the women volunteers having a better balanced diet than men. In this way, the women would be getting all the vitamins and minerals they needed from the fruit and vegetables in their regular diet.**

## Nitric Oxide Generation

**	****	****Nitric Oxide Synthase (NOS)**
**L-Arginine****	****	****Citrulline + NO****.**
**+ NADPH + O****2****	****+ NADP****+****	**
******Neuronal NOS (nNOS)**
******Inducible NOS (iNOS)**
******Endothelial NOS (eNOS)**

## NO Generation

**	****	****Nitric Oxide Synthase (NOS)**

## Reactive Nitrogen Species (RNS)

**●****NO****. ****(nitric oxide)**
******NO****2 ****(nitrogen dioxide)**
******N****2****O****3 ****(dinitrogentrioxide)**
******N****2****O****4 ****(dinitrogentetraoxide)**
******NO****2****- ****(nitrite)**
******NO****3****- ****(nitrate)**
******O=N-O-O (peroxynitrite)**
******S-nitrosothiols**
******Nitrosyl-metal complexes**

## Hypoxia inducible factor HIF-1 is stabilized by RNS or hypoxia

**●****Prolyl hydroxylase (PHD), which is inhibited by RNS, causes hydroxylation of HIF-1******
**	****at Pro****402**** or Pro****564**
******HIF-1******** binds von Hippel-Lindau protein (pVHL) and is then polyubiquitinated leading to 26S proteasome degradation**
******HIF-1******** is degraded during normoxia by the proteasome**

## RNS affect HIF and p53

**	****	****	****	****Gene activation**
**	****	****	****	****	****P300/CBP**
**RNS****	****	****HIF-1****	****	****	****	****p53****P****	****RNS**
** ****-- **
**PHD****	****	****	****	****	****	****	****   ATR/ATM**
**  ****--**
**Hypoxia****	****HIF-1-OH****	****	****	****p53****	****Hypoxia**
**VHL****	****	****	****	****	****	****	****Mdm2**
**	****	****VHL:HIF-1-OH****	****	****	****	****p53:Mdm2**
**	****	****	****Ubiquitination/26S**** **
**	****	****	****proteasome degradation**

## HIF-1 helps cells survive hypoxia

**●****As HIF-1****α**** accumulates it can form a heterodimer with HIF-1****β**** in the nucleus of the cell**
******p300/CBP binds to the HIF-1 complex altering gene expression**

## What does HIF-1 do?

**●****Helps normal tissues and tumors survive hypoxic conditions**
******Is a transcription factor which turns on over 40 genes needed for survival of hypoxic conditions**
**●****Group 1 genes such as transferrin and VEGF deliver O****2****.  Group 2 genes such as enolase 1 and hexokinase 1 provide sugars which generate energy.  Group 3 genes such as IGF-2 and IGF binding proteins 1 and 3 increase proliferation and viability.**
**●****HIF-1 increases when the O****2**** concentration is less than 4%**

## RNS affect HIF and p53

**	****	****	****	****Gene activation**
**	****	****	****	****	****P300/CBP**
**RNS****	****	****HIF-1****	****	****	****	****p53****P****	****RNS**
** ****-- **
**PHD****	****	****	****	****	****	****	****   ATR/ATM**
**  ****--**
**Hypoxia****	****HIF-1-****OH****	****	****	****p53****	****Hypoxia**
**VHL****	****	****	****	****	****	****	****Mdm2**
**	****	****VHL:HIF-1-****OH****	****	****	****	****p53:Mdm2**
**	****	****	****Ubiquitination/26S**** **
**	****	****	****proteasome degradation**

## RNS causes p53 accumulation

******Under normoxia, p53 binds to Mdm2 and is degraded**
**●****RNS down regulate Mdm2, resulting in less p53 degradation by the 26S proteasome**
******RNS causes phosphorylation of Ser****15 ****and formation of p53 tetramers. Recruitment of the co-activator p300/CBP leads to cell cycle arrest and apoptosis.**

## RNS affect HIF and p53

**	****	****	****	****Gene activation**
**	****	****	****	****	****P300/CBP**
**RNS****	****	****HIF-1****	****	****	****	****p53P****	****RNS**
** ****-- **
**PHD****	****	****	****	****	****	****	****   ****ATR/ATM**
**  ****--**
**Hypoxia****	****HIF-1-****OH****	****	****	****p53****	****Hypoxia**
**VHL****	****	****	****	****	****	****	****Mdm2**
**	****	****VHL:HIF-1-****OH****	****	****	****	****p53:Mdm2**
**	****	****	****Ubiquitination/26S**** **
**	****	****	****proteasome degradation**

## HIF-1 and p53 accumulation by RNS alters:

**●****Energy metabolism-hexokinase 1**
******Angiogenesis-VEGF**
******Iron homeostasis**
******Cell proliferation-IGF2 **
******Apoptosis and**
******Inflammation**

## Carcinogens initiate DNA damage.Initial DNA mutations lead to adenomas.  Additional mutations lead to adenocarcinomas.

## Cancer cells require the process of angiogenesis and metastasis to form distant tumors

**●****Angiogenesis-When tumors become greater than 1 mm in size, the host supplies blood vessels to deliver oxygen and nutrients required for further growth**
******Metastasis-Cancer cells can be disseminated from a primary to distant site by blood vessels or lymphatics**

## Tumors can become hypoxic without sufficient blood vessels

## Oxygen pressure is reduced in malignant relative to normal breast

## Genomic response of tumor cells to hypoxia

**●****Transcriptional activity of AP-1 (c-fos, c-jun) is increased when cancer cells are exposed to reduced oxygen pressure**
**●****NF-********B is strongly activated when cancer cells are re-exposed to normal oxygen pressure (inflammatory response)**

## Oxygen increases patient survival after radiation therapy

## HS (-) promotes the survival of neutrophils

**●****HS (-) inhibits caspase-3 cleavage and p38 MAPK phosphorylation**
*Rinaldi et al., 2006. Lab Invest. 86:391*
**	****	****	****	**

## Hydrogen sulfide

**●****The amino acids methionine and cysteine contain sulfur**
**METHIONINE****	****	****	****CYSTEINE**
**	****	****CO****2****-****	****	****	****	****	****CO****2****-**
**NH****3****+****-C-H****	****	****	****	****NH****3****+****-C-H**
**	****	****CH****2****	****	****	****	****	****CH****2**
**	****	****CH****2****	****	****	****	****	****SH**
**	****	****S**
**	****	****CH****3**

## Homocysteine is a key intermediate in sulfur metabolism

**●****Homocysteine can be metabolized under reducing conditions to methionine or oxidizing conditions to cysteine**
**	****	****	****METHIONINE****	**
**	****	****	****	****	****Methionine Synthase**
**	****	****	****HOMOCYSTEINE**
**	****	****	****	****	****Cystathionine ****β****-Synthase**
**	****	****	****CYSTATHIONINE**
**	****	****	****	****	********-Cysthathionase**
**	****	****	****CYSTEINE****	****	**

## Glutathione helps maintain cysteines in the reduced state and the iron of heme in the ferrous state (Fe2+)

**●****Glutathione is composed of glutamate, cysteine and glycine**
**	****	****	****GLUTAMATE****	****	****	****	****	****	****	********Glutamyl-cysteine Ligase**
**	****	****	********GLU-CYSTEINE**
**	****	****	****	****Glutathione Syn.**
**	****	****	********GLU-CYS-GLYCINE****	****	**

## Glutathione (GSH)

**●****is the most abundant non-protein thiol in the cell**
**●****is an antioxidant and provides cellular cytoprotection**
**●****maintains the redox environment of the cell**
**●****is increased when cells are stressed and down-regulated after a challenge has been faced.**
**	****	****	****	****	**

## Mitochondrial production and removal of superoxide

## Carbon monoxide binds to the iron in proteins which contain heme

**●****In hemoglobin C**_**=**_**O binds with higher affinity to the Fe than does O****2**** ****	****	**
**●****Carbon monoxide gas, which is released from gasoline engines, can lead to asphyxiation****	**

## CO inhibits cytochrome c oxidase

**●****CO is released when Hb is degraded**
**●****CO inhibits cytochrome c oxidase while maintaining cellular ATP levels resulting in the generation of ROS ****	**
**●****CO addition to cells resulted in p38 MAPK phosphorylation**
*Zuckerbraun et al (2007), FASEB J 21:1099***	**

## CO levels are reduced by heme oxygenase inhibitors

**●****Heme****	****	****CO **
**	****	****Heme-oxygenase (HO)**
**●****HO is inhibited by imidazoles or metalloporphyrins**
*Kinode et al., Can. J Physiol. Pharmacol.  (2008), 86: 577.***	**

## Redox Species which may alter carcinogenesis 

******Reactive oxygen species (ROS)**
**●****Reactive nitrogen species (RNS)**
******Hydrogen sulfide**
******Carbon monoxide**

## References

**● ****Zhou, C.G. and Banerjee, R. (2005), Homocysteine and redox signaling. Antioxidants & Redox Signaling 7:547-59.**
**●****Zhou, J, Schmid, T. and Brune, B. (2004), HIF-1****α**** and p53 as targets of NO in affecting cell proliferation, death and adaptation.  Current Mol. Med. 4:741-51.**