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Site Directed Mutagenesis and Protein Engineering

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Title: Site Directed Mutagenesis and Protein Engineering


1
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

2
Overview
3
Breast cancer
  • According to the globocan-2008 report, incidence
    for breast cancer in Iran was 18.4 per100,000
    person-years.

4
OXIDATIVE STRESS CANCER
5
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)

6
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

7
Oxidation/reduction balance
8
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

9
HMOX-1
10
HMOX-1
11
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

12
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

13
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.
14
Schematic model of Nrf2 regulation by Keap1
15
Nrf2 in cancer promotion
Dark side of Nrf2
16
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

17
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

18
(No Transcript)
19
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

20
Effect of ascorbic acid on Nrf2
21
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

22
Methods Materials
Biochemical Assays
3
Western blotting metod
2
Cell culture procedures
1
23
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

24
2. Western blotting
  • Primary antibody
  • Nrf2 Antibody (C-20) sc-722
  • Nrf2 Antibody (H-300) sc-13032
  • Secondry antibody

25
3. Biochemical Assays
  • Measurement of antioxidant enzyms
  • Heme Oxygenase 1 (HMOX-1) Motterlini.R method
  • NAD (P) H quinone oxidoreductase 1 (NQO1)
    Ernester method
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