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- constantly renewed
Different cells within the
intestine
Stem cells: Their role in cancer
(CSC)
They have ability of self-renewal and are
sufficiently long-living to receive mutations
leading to cancer
2 pathways:
◦ Planar: Ca2+ involved, contols cellular movement and
polarity
◦ Canonical: β-catenin involved, regulates cell
proliferation
Canonical Wnt pathway
APC: A critical protein in colorectal cancer
Its role in the Wnt pathway
APC = Adenomatous
polyposis coli protein
WT APC
C-
terminally
truncated
APC
Mutations that are necessary for
the development of colorectal
cancer
From mutation in stem cells to
colorectal cancer
•the “bottom-up”
model
Bottom-up Top-down
model model
From mutation in stem cells to
colorectal cancer
•Formation of
a monocryptal
adenoma
Vitamin A and D
Small-molecule inhibitors
Antibodies
Non-steroidal anti-inflammatory
drugs (NSAIDS)
e.g. aspirin, sulindac and indomethacin
Effects:
Inhibiting proliferation
Inducing apoptosis
Curbing cancer cell invasion
Transmembrane protein
Functions:
◦ surface adhesion
◦ Mediates apoptosis resistance
◦ growthfactor/signal transduction
Adenoviral virion (Ad5)
•non enveloped icosahedral “particle”
4
Entry through cancer-cell-
specific receptors
1. First step: retargeting
Synthetic promoter
High specificity
regulated beta-catenin
Different strategies
siRNA repressing an anti-
apoptotic gene, like Bcl2
M protein expression
Choice of insert
Vesicular stomatitis
virus (VSV):
• negative-stranded
RNA virus
• infects mammals
• kills tumor cells
830 bp mRNA
encodes M protein of
229 aa
M protein
Induces apoptosis in 2 ways:
• Activates caspase 9
• Inhibits host RNA polymerase I , II, III
Inhibits nuclear-cytoplasmic transport of RNA =>
decrease of transcription initiation factors in
cytoplasm
Structure
1. Theory part
I. Introduction: epidemology, CSC
II. Wnt pathway and the development of colon cancer
III. Drug development: problems and possibilities
2. Research part
I. Virus-based approach
II. Validation
III. Therapy design and side effects
IV. Personalized therapy
Validation and search for the
best dosage
Expressionof M protein in infected
tumor culture
Specificity of infection and expression
Stop of cell proliferation
Induction of apoptosis
…
Expression of the M protein and
its specifity to colon cancer
tissue Procedure
• culture of normal colon cells and tumor colon cells
• infection with virus expressing M protein construct
• purify the protein fraction from the cell samples
•Immunoblot with specific anti-M protein antibody
Immunoblot
Infection
Protein
extraction
M M
Control Tumor
Expression of the M protein and
its specifity to colon cancer
tissue Procedure
• culture of normal colon cells and tumor colon cells
• infection with virus expressing M protein construct
• purify the protein fraction from the cell samples
•Immunoblot with specific anti-M protein antibody
Immunoblot
Infection
Protein
extraction
M M
Control Tumor
Expression of the M protein and
its specifity to colon cancer
tissue Procedure
• culture of normal colon cells and tumor colon cells
• infection with virus expressing M protein construct
• purify the protein fraction from the cell samples
•Immunoblot with specific anti-M protein antibody
Immunoblot
Infection
Protein
extraction
M M
Control Tumor
Expression of the M protein and
its specifity to colon cancer
tissue Procedure
• culture of normal colon cells and tumor colon cells
• infection with virus expressing M protein construct
• purify the protein fraction from the cell samples
•Immunoblot with specific anti-M protein antibody
Immunoblot
Infection
Protein
extraction
M M
Control Tumor
Detection of cell proliferation in
target cells
CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS)
Procedure
•tissue culture, plating in 96-well plate
•infect with virus, use different dosages
−expressing M protein or PBS
•add MTS
•read absorbtion at 490nm
Detection of apoptosis in target
cells
Mito CaptureTM Apoptosis Detection Kit
Cationic dye
Healthy cells red fluorescence
Apoptotic cells green fluorescence
Detection: fluorescence microscopy or flow cytometer
Procedure
•cell culture
•infect with virus, use different
dosages
−expressing M protein or PBS
•staining
•qualitative test: microscope
•quantitative test: flow cytometer
Structure
1. Theory part
I. Introduction: epidemology, CSC
II. Wnt pathway and the development of colon cancer
III. Drug development: problems and possibilities
2. Research part
I. Virus-based approach
II. Validation
III. Therapy design and side effects
IV. Personalized therapy
Therapy design: Method of
delivery
Possibilities:
Intravenous injection
◦ Systemic distribution: Elevated risk of side
effects
◦ Non-homogenous distribution in tumor
Intratumoral implantation
◦ Elevated risk of immune response
Intratumoral injection
◦ More specific targeting
◦ Risks of systemic distribution minimized
Non-replicating virus in normal cells
CD44 restriction
(PEG)
Therapy design: Method of
delivery
Possibilities:
Intravenous injection
◦ Systemic distribution: Elevated risk of side
effects
◦ Non-homogenous distribution in tumor
Intratumoral implantation
◦ Elevated risk of immune response
Intratumoral injection
◦ More specific targeting
◦ Risks of systemic distribution minimized
Non-replicating virus in normal cells
CD44 restriction
(PEG)
Shielding?
Aim:
◦ Evade neutralizing antibodies
◦ Lower clearance ratio
◦ Block transduction to liver
◦ Easier storage
Virus:
• Not replicating in normal cells
• CD44 restriction
• CTP4: specific promoter
• (PEG)