1. Hypothesis
Inhibi.on
of
HMOX-­‐1
through
ShRNA
will
lead
to
an
increased
effec.veness
of
gemcitabine
and
thereby
augment
cancer
cell
death.
Similarly,
overexpression
of
HMOX-­‐1
through
the
same
method
will
lead
to
a
decrease
in
effec.veness
and
lower
death
count
of
cancer
cells.
Experimental
Method
PI
staining
gives
the
advantage
of
dis.nguishing
between
living
and
dead
cells,
as
DNA
of
dead
cells
will
be
fluorescent
and
living
ones
will
not.
Also,
it
allows
us
to
use
flow
cytometry
to
analyze
the
cell
count
based
on
their
physical
and
chemical
characteris.cs.
Introduc.on
One
in
nine
Canadian
women
is
expected
to
develop
breast
cancer
in
her
life.me
(1).
Heme-­‐oxygenase-­‐1
(HMOX-­‐1)
is
an
enzyme
that
degrades
pro-­‐oxidant
heme,
an
iron
containing
compound
present
in
hemoglobin,
and
other
hemoproteins.
The
products
of
this
reac.on
have
an.oxidant
proper.es,
which
protect
cell
membranes
from
reac.ve
oxygen
species.
This
effect
can
be
exploited
by
cancerous
cells
into
protec.ng
themselves
against
chemotherapy
drugs
such
as
gemcitabine,
which
cause
oxida.ve
stress
to
cells.
Ra.onale
An.cipated
Results
Assuming
HMOX-­‐1
decreases
the
effec.veness
of
gemcitabine
treatment
in
4T1
mouse
breast
cancer
cells,
the
sample
with
HMOX-­‐1
knockdown
would
have
the
highest
count
of
fluorescent
(dead)
cells
present,
followed
by
the
controlled
sample.
Lastly,
the
sample
with
HMOX-­‐1
overexpression
would
give
the
lowest
count
of
fluorescent
cells,
indica.ng
reduced
efficiency
of
gemcitabine
in
trea.ng
breast
cancer.
Discussion
The
experiment
will
be
carried
in
vitro,
and
past
experiments
have
shown
that
HMOX-­‐1
may
behave
differently
in
vivo
than
it
does
in
vitro
(4,7).
This
suggests
that
further
experiments
should
be
carried
out
researching
the
difference
in
vivo.
If
the
results
of
this
experiment
are
conclusive,
then
the
informa.on
could
be
applicable
to
other
chemotherapy
agents
or
other
types
of
cancer.
In
addi.on,
HMOX-­‐1
chemical
inhibitors,
such
as
zinc
or
.n
protoporphyrin
(ZnPPIX,
or
SnPPIX),
could
be
used
along
with
gemcitabine
or
other
chemotherapy
agents
to
work
synergis.cally
and
produce
growth
inhibi.on
in
gemcitabine-­‐resistant
breast
cancer
cells.
References
(1)
Canadian
breast
cancer
founda.on.
(2013).
Retrieved
from
hYps://www.cbcf.org/central/Pages/default.aspx.
(2)
Lau,
Alexandria,
Nicole
V.,
Zheng
S.,
Pak
K.,
and
Donna
D.
"Dual
Roles
of
Nrf2
in
Cancer."
Pharmacological
Research
58.5-­‐6
(2008):
262-­‐70
(3)
Nuhn,
P.,
Kunzli,
B.,
&
Hennig,
R.,et
al
(2009).
Heme
oxygenase-­‐1
and
its
metabolites
affect
pancrea.c
tumor
growth
in
vivo.
Mol
Cancer,
8(7),
37-­‐43.
(4)Was,
H.,
Jozef,
D.,
&
Alicja,
J.
(2012).
Heme
oxygenase-­‐1
in
tumor
biology
and
therapy.
Current
Drug
Targets,
11(12),
1551-­‐1570.
(5)
Nowis,
D.,
et.al06).
Heme
oxygenase-­‐1
protects
tumor
cells
against
photodynamic
therapy-­‐mediated
cytotoxicity.
Oncogene,
25(24),
3365-­‐3374.
(6)
Berberat,
P.,
et.al
(2005).
Inhibi.on
of
heme
oxygenase-­‐1
increases
responsiveness
of
pancrea.c
cancer
cells
to
an.cancer
treatment.
Clinical
Cancer
Research,
10(11),
3790.
(7)
Ryter,
S.,
Alam,
J.,
&
Choi,
A.
(2006).
Heme
oxygenase-­‐1/carbon
monoxide:
From
basic
science
to
therapeu.c
applica.ons.
Physiological
Reviews,
86(85),
583-­‐650.
(8)
Song
W,
Su
H,
Song
S,
Paudel
HK,
Schipper
HM.
Over-­‐expression
of
heme
oxygenase-­‐1
promotes
oxida.ve
mitochondrial
damage
in
rat
astroglia.
J
Cell
Physiol
2006;
206:
655-­‐63.
Image
(a)DNA
photo:
hYp://watchdog.wpengine.netdna-­‐cdn.com/wp-­‐content/blogs.dir/1/files/2013/11/shuYerstock_61775431.jpg
Image
(b)
Petri
dish
photo:
hYp://www.clker.com/clipart-­‐342081.html
Figure
1
Kim,
Ada.
Unpublished
data.
HMOX-­‐1
knocked
down
Murine
mammary
4T1
carcinoma
cells
Inhibi.on
by
shRNA
Overexpression
by
an
expression
vector
HMOX-­‐1
overexpressed
Control
Chemotherapy
agents
such
as
gemcitabine
are
stress
factors
for
cancer
cells.
HMOX-­‐1
is
a
key
enzyme
for
protec.on
against
oxida.ve
stress,
providing
resistance
to
chemotherapy
(6,7)
Inhibi.ng
HMOX-­‐1
will
lower
the
defense
of
cancer
cells,
making
gemcitabine
more
effec.ve
(8)
Inhibited
Control
Overexpressed
Arbitrary
Number
Living
Cells
A4er
Gemcitabine
Treatment
Acknowledgement
We
would
like
to
thank
Ada
Kim-­‐
our
mentor,
and
the
URO
team
for
all
the
help
they
have
provided.
Figure
1.
Hypothesized
Results:
Higher
number
of
living
cancer
cells
amer
treatment
with
gemcitabine
in
the
overexpressed
group,
and
lowest
in
the
inhibited
group
Treat
the
cell
cultures
with
gemcitabine
Analyze
viability
using
Propidium
Iodide
(PI)
staining
and
flow
cytometry
Analyze
cell
cultures
for
presence
or
absence
of
HMOX-­‐1
using
Western
Blonng
Image
(a)
Image
(b)
heme
+
NAD(P)H
+
H+
+
3
O2
↔
biliverdin
+
Fe2+
+
CO
+
NAD(P)+
+
3
H2O
(2)
Figure
1b:
Western
Blot
showing
inhibi.on
of
HMOX-­‐1
in
4T1
murine
mammary
carcinoma
cells
Figure
1a:
Western
Blot
showing
overexpression
of
HMOX-­‐1
in
4T1
murine
mammary
carcinoma
cells
Previous
Research
This
rela.onship
has
been
studied
with
regards
to
some
types
of
cancer:
high
HMOX1
expression
in
pancrea.c
cancer
cell
lines
was
associated
with
increased
chemoresistance
to
gemcitabine
(3),
but
other
studies
have
shown
that
inhibi.on
of
HMOX-­‐1
does
not
necessarily
lead
to
increased
effec.veness
of
treatment
in
all
types
of
cancer(4,5).
However,
the
cytoprotec.ve
role
of
HMOX1
amer
chemotherapeu.c
treatment
is
unknown
in
breast
cancer.