Site Directed Mutagenesis and Protein Engineering

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The assessment of HMOX-1 and NQO1 Enzyme activity
and the level of Nrf2 protein in breast cancer
cell lines treated with chemopreventive agents

• Supervisor
• Dr. Z. Mostafavi-Pour
• Presented by
• F. Keshavarzi

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Overview
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Breast cancer

• According to the globocan-2008 report, incidence
  for breast cancer in Iran was 18.4 per100,000
  person-years.

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OXIDATIVE STRESS CANCER
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Reactive oxygen species (ROS)

• ROS are reactive, strongly oxidizing forms of
  oxygen species
• ROS are produced naturally and continuously
  within the cell
• From our O2 consumption
• 90 is committed to oxidative phosphorylation
• 10 is used by enzymes responsible for
  hydroxylation and oxygenation reactions
• lt1 is converted to reactive oxygen species (ROS)

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ROS cont

• ROS are physiologically important in
• Regulation of metabolism (some enzymes use H2O2
  as a substrate)
• Immunologic defenses against infection
• ROS overproduction can cause oxidative damage to
  macromolecules such as
• Protein thiol groups, DNA bases and
  Polyunsaturated fatty acids (PUFA)
• Oxidative stress can affect numerous cellular
  processes including
• DNA repair
• Proliferation
• Migration
• Cellular adhesion

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Oxidation/reduction balance
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HMOX-1

• Is a member of the heat shock protein family
• Its expression is mainly upregulated by Nrf2
• Plays a pivotal role in the maintenance of
  cellular redox homeostasis
• Prevents transformation of normal cells to
  cancerous by counteracting ROS-mediated
  carcinogenesis
• Is the rate limiting enzyme in the degradation of
  heme into biliverdin, carbon monoxide (CO), and
  free iron
• Its role as an effective antioxidant is
  significantly different

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HMOX-1
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HMOX-1
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NQO1

• Also known as DT-diaphorase, menadione reductase,
  or quinone reductase 1
• A cytoplasmic flavoenzyme encoded by a gene
  located on chromosome 16q22
• Uses NADH or NADPH as substrates to directly
  reduce quinones to hydroquinones
• Is a highly inducible enzyme that is regulated by
  the Keap1/Nrf2/ARE pathway

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NQO1

• Functions of NQO1 include
• Xenobiotic detoxification,
• Superoxide scavenging
• The maintenance of endogenous antioxidant
  vitamins
• Shown to be identical to the dicoumarol-inhibited
  vitamin K reductase described by Märki and
  Martius
• Binds to and thereby stabilizes the important
  tumor suppressor p53 against proteasomal
  degradation

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gene Nuclear factor (erythroid-derived 2)-like 2
(Nrf2)

• Is a member of the cap n collar (CNC) subfamily
  of transcription factors
• Contains a basic leucine zipper DNA binding
  domain (bZip) at the C-terminus
• Was found to be ubiquitiously expressed in many
  organs

Nrf2 signaling and cell survival. Suryakant K.
Niture, James W. Kaspar, Jun Shen, Anil K.
Jaiswal. Toxicology and Applied Pharmacology 244
(2010) 3742.
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Schematic model of Nrf2 regulation by Keap1
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Nrf2 in cancer promotion
Dark side of Nrf2
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HMOX-1 as a promising marker

• HMOX-1 Gene is upregulated in tumors, compared
  with surrounding healthy tissues, confer a great
  advantage to cancer cells for survival against
  anti-cancer drugs and irradiation
• The levels of nuclear HMOX-1 in the tumor
  specimens were higher than those in nonmalignant
  tissues, which appears to be associated with
  tumor progression
• HMOX-1 as a promising marker for the diagnosis of
  cancers

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NQO1 as a promising marker

• NQO1 Gene is upregulated in tumors, compared with
  surrounding healthy tissues, confer a great
  advantage to cancer cells for survival against
  anti-cancer drugs and irradiation
• High-level expression of NQO1 may be a potential
  biomarker for poor prognostic evaluation of
  cancers

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Quercetin

• Quercetin (3,3,4,5,7-pentahydroxyflavone) is a
  plant derived flavonoid
• Exert protective effects against hydrogen
  peroxide-induced cytotoxicity 1025 µM in rat
  hepatoma cells.
• Induced cytotoxicity, DNA strand break,
  oligonucleosomal DNA fragmentation, and caspase
  activation at 50 and 250 µM
• Nrf2 is activated in response to mild and acute
  quercetin treatment as an adaptive response to
  guard against oxidative and inflammatory cell
  damage, but Nrf2 might not play a protective role
  against long and cytotoxic flavonoid exposure

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Effect of ascorbic acid on Nrf2
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Goal Objectives

• Goal
• To decrease enzymatic activity of NQO1 and
  HMOX-1 biomarkers and nrf2 protein expresion in
  cancer cell lines
• Objectives
• Determination of the effect of quercetin
  vitamin C combination treatment on Nrf2 protein
  expression
• Measurement the activity of HMOX-1 in breast
  cancer cell lines
• Measurement the activity of NQO1 in breast cancer
  cell lines

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Methods Materials
Biochemical Assays
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Western blotting metod
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Cell culture procedures
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1. Cell culture procedures

• Culture of MDA-MB-468, MDA-MB-231, MCF-7 A549
  in RPMI1640 media
• Treatment of cells with 75µM Quercetin 100 µM
  Vitamin C

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2. Western blotting

• Primary antibody
• Nrf2 Antibody (C-20) sc-722
• Nrf2 Antibody (H-300) sc-13032
• Secondry antibody

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3. Biochemical Assays

• Measurement of antioxidant enzyms
• Heme Oxygenase 1 (HMOX-1) Motterlini.R method
• NAD (P) H quinone oxidoreductase 1 (NQO1)
  Ernester method