Download Fas-associating death domain protein overexpression

Fas-associating death domain protein overexpression
induces apoptosis in lung cancer cells
20641001 Kyung hye Koh
Contents
OUTLINE
INTRODUCTION
MATERIALS & METHODS
RESULTS
DISCUSSION
IMPRESSION
Outline
overexpression induces
Conclusion FADD
apoptosis in lung cancer cells
Results
Increasing multiplicities of infection
Methods
Objectives
Adenoviral vector/Immunoblotting analysis
Apoptosis via FADD overexpression, novel therapy in
pulmonary neoplasms
Introduction
• 14% 5-year survival rate of patients with
non-small cell lung cancer
• Fas-associating death domain protein/ mediator
of receptor-induced toxicity (FADD/MORT1)
• 29 kD cytoplasmic adaptor molecule , mediates
signal transduction pathways of apoptosis
• Overexpression of intracellular FADD would
induce caspase-8 activation and apoptosis
Materials & Methods
Cells and reagents
• A549 (alveolar cell carcinoma)
• NCI-H226 (squamous cell carcinoma) Both
maintained in media
Viral Vectors and Infection
• XhoI/XhoI 720 bp murine FADD cDNA coding
regioninserted to create Ad-FADD expression
Vector
• Ad-EGFP (enhanced green fluorescent protein)
Materials & Methods
Immunoblotting Analysis
• 13% SDS PAGE transferred onto nitrocellulose
membrane
• Incubated with anti-FADD, anti-caspase-8 or
anti-α-actin antibodies
Cell Viability Assays
• Determined by crystal violet method
• Calculated from relative dye intensity
Materials & Methods
Caspase Activity Assay
• Using fluorometric change upon cleavage of a
carboxyfluorescene-labeled fluoromethyl
ketone-peptide inhibitor (VAD)
Statistical Analysis
• One-way analysis of variance (ANOVA) by use
of the Student-Newman-Keuls method
Results
FADD Expression in A549 Lung Cancer Cells
•A549 cells infected with
Ad-FADD in increasing
concentrations
• Results presented by
western blotting
•Actin levels confirm equal
loading of protein in each
lane
Dose-dependent increase in FADD expression in A549
lung cancer cells
Results
Ad-FADD Infection and Dose-Dependent Apoptotic
Cell Death
•Cell structure was photographed
under phase-contrast microscopy
• Apoptotic morphology consisting
of detached,condensed cells with
membrane blebbing
Increasing doses of Ad- FADD (B to E) resulted in augmented
numbers of apoptotic cells
Results
Ad-FADD Infection and Dose-Dependent Apoptotic
Cell Death
•To quantitate cell death
induced by Ad-FADD in
A549 cells
• Cells were fixed and
stained with crystal violet,
lysed, and quantitated
by spectrophotometry at
550 nm
Ad-FADD infection induced a dose-dependent increase in
cell death compared with Ad-EGFP infection
Results
Ad-FADD Treatment- and Concentration Dependent
Caspase-8 Cleavage
•A549 cells were plated in
6-well dishes and infected
with increasing multiplicities
of infection of Ad-FADD
(MOI: 0, 5, 10, 25, and 50)
• Expression levels of
cleaved and uncleaved
forms of caspase-8 were
assessed by Western
blotting
Ad-FADD resulted in dose-dependent elevations in active
caspase-8, with corresponding reductions in expression
of the procaspase-8 zymogen
Results
Ad-FADD Infection-Induced Apoptotic Cell Death
and Caspase Activation in NCI-H226 Lung Cancer
Cells
• NCI-H226 cells infected with
Ad-FADD
•Cell viability was determined
at 24 hours by crystal violet
staining
• Caspase activity was
assessed by fluorescently
labelled z-VAD (red, arrows)
and Hoescht for nuclei (blue)
Ad-FADD infection induces apoptotic cell death in NCIH226 cells
Discussion
• In p53-deficient cells or cells that overexpress
Bcl-2, apoptosis could still be induced by Fas
ligand
• The Mutations in p53 are the most common
mutation in human cancer
• The overexpression of FADD may be a viable
alternative to other therapeutic modalities that
require wild-type p53
Discussion
• The Overexpression of FADD alone could
induce apoptotic cell death in human lung cancer
cells
Impression
• To induce apoptosis via FADD overexpression
at cancer cells, production of cancer-specific
vector that encodes fadd must be preceded
• Lung cancer cell death via overexpression of
FADD may be enhanced in combination with
Chemotherapy and radiation
Impression
• In addition, as adenoviral vectors are rapidly
cleared by the immune system, ‘bystander effect’
should be induced in intratumoral injection
• Except using adenoviral vector, researches
related to other methods to increase Endogenous
FADD expression should be investigated