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Headquarters of the

Electron Microscope Unit Australian Microscopy & Microanalysis Research Facility


The University of Sydney Australian Key Centre for Microscopy and Microanalysis
Australia ARC Centre of Excellence for Design in Light Metals

Inhibition of histone deacetylases reduces the expression of ROCK 1 in high-density


collagen matrix by indirect mechanisms
Vanisri Raviraj1*, Eric Thompson2, Lilian Soon1*
1
AKCMM, University of Sydney, Sydney, NSW, Australia, 2Department of Surgery, St Vincent’s Institute, Melbourne, VIC, Australia
*v.raviraj@usyd.edu.au, *l.soon@usyd.edu.au

Introduction Methodology
Rat breast adenocarcinoma cells, MTLn3 cells were grown on high-density (20 mg/ml) 3D collagen matrix in complete AdvDMEM without
In cancer, reduced histone acetylation is significantly correlated
antibiotics. Twenty four hour after seeding, cells were treated with different concentration of MS-275 alone or combination with other drugs for
with advanced tumor stages and depth of the cancer invasion
(1). Histone deacetylases (HDACs) have been recently shown to 48hr.
be important factors in cell migration and invasion for both normal Confocal microscopy: forty-eight hours after MS-275 treatment, cells were fixed in 4% PFA and stained in Alexa fluor 488 phalloidin. Cells were
and malignant cells. HDAC inhibitors are being used in clinical Imaged under the Olympus FV1000 confocal microscope. Live cell images were taken in GFP-actin transfected cells.
trails to counter cancer and they function by blocking cell Extraction of total RNA and Q-PCR: Total RNA was extracted using Trizol and RNeasy Mini extraction kit. One micro gram of total RNA was
proliferation, angiogenesis and cell invasion and by promoting reverse transcribed using Superscript111 First strand synthesis system and 1µl of cDNA was used to quantify genes in Rotor gene 6000.
cell differentiation and apoptosis (2). In our study, the class I Protein extraction and ROCK activity assay : HD gels were digested in 0.5mg/ml Collagenase, supplemented with 1X Kerb’s ringer buffer and
HDAC inhibitor, MS-275, significantly downregulates the 100mM CaCl2 for 30min at 37oC. Cells were pelleted at 2000rpm and washed 2 times in cold PBS. Cells were lysed in RIPA buffer and
metastasis promoter gene ROCK1 in mRNA, protein level and
supernatant was separated at 13,000rpm for 15min. Ten microgram of protein was used to measure activity using ROCK activity assay kit from
activity levels. This effect is mediated by intermediate molecules
(p53 and Notch). Cell Biolabs Inc. In Brief, 10µg of protein was incubated in 65ul of 1X Kinase/ ATP/ ROCK substrate solution (MYPT1) at 37oC for 30min.
Protein/substrate was separated 12% SDS gels.

Results Qui ckT im e ª a nd a


de co m pre ss or
a re ne ed ed t o s ee t his p ict ur e.

1 µM 3 µM RT-PCR for N-WASP RT-PCR for RhoB


Control
0’ 10’ 12’ 14’ MS-275 MS-275 2
1.8
8
7
1.6 6
1.4
1.2 5
QuickTimeª and a QuickTimeª and a WASP1 4
are needed to see thisTop ofthe
decompressor decompressor
are needed to see this picture. picture. 0.8
Relative N- 3
0.6
collagen gel 0.4
2
1
0.2 expression
0
QuickTimeª and a
decompressor
are needed to see this picture. 0 Relative RhoB
Control 1uM MS- 3uM MS- Control 1uM MS- 3uM MS-
275 275 275 275
Q u ickTim eª and a QuickTim eª and a
Inside the
d ecom p ressor
are nee ded to se e thisarepictu
decom pressor
re.to see this picture.
needed collagen gel Con 1µM 3 µM
N-WASP a
ren
eed
edt
o s
ee
Q
u

t
h
ick
d
is
T
e
p
im
c
o
ic
t
e
m
u
ªa

r
e
p
.
n
r
e
d
s
a
s
or

α -tubulin
Quick
T im
e ªanda
decom pre
sso r
a
ren
eed
edtos
e ethispic
ture.

MS-275 increased the number and size of MS-275 increased the expression of RhoB and N-WASP,
Live cell imaging showing protrusion- and
protrusions in MTLn3 cells. genes that function in cell migration.
contractility-led invasion mechanisms.

RT-PCR for ROCK1


RT-PCR for ROCK1 RT-PCR for ROCK1
1.40 1µM 3µM 1.4 1.2
MS-275 suppress
1.20
1.00
Control MS-275 MS-275 1.2
1 the ROCK1
ROCK1 1
indirectly because
QuickTim e
ªa nda

0.80 0.8
deco mp ress
o r
a
ren
eed
edtos
e eth ispictu
re.

0.60
0.8
HD LD
0.40
Phospho Q u
i
ckT
d
i
e
m
c
o
e
m
ªa
n
p
r
d
e
s
a
s
or
0.6 Phospho- 0.6 inhibition with
-MYPT1
a
ren
ee
de
dt
o s
eet
hisp
ict
ure.
Q u
ick Timeªand a

MYPT1(T696 )
d ec o
m pr
e s
s o
r
0.4 0.4
cycloheximide
a
rene
ede
d t
o s
ee t
h ispic
ture.

0.20 expression
expression
0.00 α -tubulin
0.2
Relative ROCK1 α -tubulin
Quic
k
d
T
ec
im
o
m
eªa
p r
e
nda
s
so r
0.2
(CHX) which blocks
a
ren
eed
edtos
eeth ispicture.

Relative ROCK1
Q

0
expression
a

Control 1uM MS- 3uM MS- 0


r

u
e

HD(20mg/ml) LD(1mg/ml)
e

Relative ROCK1 protein expression,


i
c

275 275
c
o
n

275 275
m

reversed the down-


e

CHX
e

i
p

m
d

ROCK transcript and activity levels are highly up- regulation of


MS-275
r

MS-275 down-regulates ROCK1 mRNA and kinase Control


1uM MS-3uM MS-10ug/ml
e

e
e
d

ª
s

regulated in HD matrix. CHX+3uM


ROCK1 by MS-275.
activity in HD matrix.
s

a
t

o
o

n
r

d
s
e

a
e
t
h

RT-PCR for Notch1 RT-PCR for


i
s

RT-PCR for ROCK1


p53
p

1.8 1.6
i
c

1.6 1.4 1.4 The up-regulation of


t
u

1.4 1.2 1.2


Notch1 and p53 by
r

1
e

1.2 1
.

Notch1
1 3uM0.8
Relative 0.8
0.6 MS-275 is responsible
0.8 0.6 MS-275
0.4
0.6 0.4
expression 0.2 for the inhibition of
0.4 Relative
0.2 p53 expression
0.2
0
0
Relative ROCK1
ROCK1; drug-ablation
0
Control 1uMMS- 3uMMS- of p53 or Notch1
Control 1uM MS- 3uM MS-
275 275 275 275 Control reversed the effects of
3uMMS-275 10uMDAPT MS-275 on ROCK1
10uMPifithrin MS-275&DAPT
MS-275 increased the transcripts of the tumour suppressors, Notch1 and p53, in MS-275&Pifithrin levels.
concentration dependant manner.

Summary References Acknowledgement


1. Tao Liu, S. K., Andrew Tee, Glenn M. Marshall
The micro-environment is a regulator of the expression of ROCK, which was up-regulated This study has been supported by the NHMRC
(2006). "Histone deacetylase inhibitors:
in HD matrix compared to LD matrix. MS-275 down-regulated ROCK1 in mRNA, protein (# 402510) and ARC (DP0881012). Vanisri
Multifunctional anticancer agents." Cancer treatment
and activity levels. Cycloheximide reversed ROCK1 suppression by MS-275 suggesting Raviraj is funded on a NHMRC scholarship.
reviews 32: 157-165.
that the effects of MS-275 on ROCK1 expression is indirect. Notch1 and p53 were directly We also gratefully acknowledge the Australian
2. Yasui W, O. N., Ono S, Mitani Y, Ito R, Nakayama H
up-regulated by MS-275 and were identified as the intermediaries in controlling ROCK1 Microscopy and Microanalysis Research
(2003). "Histone acetylation and gastrointestinal
levels in cells treated with MS-275. Facility (AMMRF), AKCMM, Sydney, Australia
carcinogenesis." Ann N Y Acad Sci 983.
for staff assistance and utilization of the facility.