The early testosterone-induced actin reorganization in colon cancer is
independent of iAR signalling
Saad Alkahtani
Department of Biology, Teachers College, King Saud University, Riyadh, Saudi Arabia.
A
15min
control
30min
60min
+ TAC
15min
control
G
G/ F ratio %
Summary:
Recently it has been reported that testosterone
conjugates trigger rapid actin cytoskeleton
reorganization in colon tumour cells. This effect
was recognized as a critical initial event, controlling
apoptosis and inhibiting cell migration. The present
study addressed whether these early effects in
colon tumours are controlled by the classical
androgen receptor (iAR) and/or through the
activation of the newly identified membrane
androgen receptors (mAR). In line with previous
observations, treatment of Caco2 colon cancer cells
with testosterone albumin conjugates (TAC)
induced rapid (within 15 minutes) actin
cytoskeleton restructuring. This was evident from
confocal laser microscopic analysis showing clear
formation of newly synthesized actin filaments,
while quantitative determinations of the G to Factin ratio fully confirmed the rapid actin
polymerization. The same experiments were then
performed in the presence of the specific
nonsteroidal antiandrogen flutamide or in cells
transfected with a specific siRNA targeting iAR that
resulted in reduced expression of iAR. Upon
blocking of iAR either by flutamide or by siRNA
silencing actin polymerization was still induced in
Caco2 cells. These experiments underlined that the
rapid testosterone-induced actin polymerization is
not correlated with the classical androgen receptor
signalling and imply the involvement of early mAR
signalling in testosterone-treated colon cancer
cells.
B
+ TAC
F
G
80
F
G
F
60min
G
* **
60
20
F
n=5
40
control 15 min 30 min 60 min
Fig.1. (A) 24h serum-starved cells were stimulated with 10-7 M testosterone-HSA. Cells were stained with rhodamine-phalloidin for filamentous actin
and DRAQ5™ for nuclei and subsequently analysed by confocal laser scanning microscopy . (B) G- and F- actin were measured by quantitative
immunoblot analysis after Triton X-100 subcellular fractionation. Bars present the G/F actin mean value  SE of n=5 independent duplicate experiments
(** P < 0.01, * P<0.05).
iAR- siRNA + TAC
Flutamide + TAC
control
control
15min
30min
60min
flutamide+TAC
30min
15min
control
15min
control
60min
30min
60min
neg
siRNA
iAR
siRNA
iAR
fold decrease
G
Introduction:
Early actin cytoskeleton reorganization is an
important initial step of the non-genomic steroid
hormone actions of androgens in a variety of tumor
cell models [1-3]. Actin restructuring modifies cellsubstratum adhesion and has been reported to
control many aspects of cellular physiology,
including cell motility, secretion, apoptosis, cell
proliferation and survival.From these and many
other reports it became widely accepted that
modifications in microfilament dynamics triggered
by membrane receptor signaling play a pivotal role
in various cell responses and seem to be involved in
a large number of human diseases [4]. However,
the potential involvement of the classical androgen
receptor (iAR) in the early actin reorganization has
not been addressed. In the present study we have
analyzed the rapid actin restructuring in the
presence of the specific nonsteroidal antiandrogen
flutamide and cells transfected with a specific
siRNA targeting iAR that resulted in reduced
expression of iAR. Our results indicate specific and
rapid testosterone effects on actin reorganiztion
that are independent of iAR signaling.
30min
F
G
F
G
F
G
F
Fig.2. 24h serum-starved cells were stimulated with 10-7 M testosteroneHSA in the presence or absence of 10-6 M flutamide. Cells were stained
with rhodamine-phalloidin for filamentous actin and DRAQ5™ for nuclei
and subsequently analysed by confocal laser scanning microscopy .
control
TAC-FITC
Flutamide treated Caco-2 cells
TAC-FITC
HSA-FITC
iAR silenced Caco-2 cells
TAC-FITC
HSA-FITC
Fig.4. Confocal laser scanning microscopy of Caco-2 cells treated with
either 10-6 M flutamide (left panels) or iAR siRNA (right panels) and
stained with testosterone-HSA-FITC. Magnification ×100.
.
1.0
0.87±0.11
0.28±0.02 **
actin
n=3
Fig.3. 24h serum-starved cells were stimulated with 10-7 M testosteroneHSA were silenced with or without siRNA against iAR for the indicated
time. Cells were stained with rhodamine-phalloidin for filamentous actin
and DRAQ5™ for nuclei and subsequently analysed by confocal laser
scanning microscopy .
Results:
In line with previous observations [1], treatment of Caco2 colon tumor
cells with testosterone albumin conjugates (TAC) induced rapid (within
15 minutes) actin cytoskeleton restructuring. This was evident from
confocal laser microscopic analysis showing clear formation of newly
synthesized actin filaments (Fig. 1 A), while quantitative determinations
of the G to F-actin ratio fully confirmed the rapid actin polymerization
(Fig. 1B). To identify the potential involvement of the classical androgen
receptor (iAR) in this early event, the same experiments were performed
in the presence of the specific nonsteroidal antiandrogen flutamide (Fig
2), or in cells transfected with a specific siRNA targeting iAR that resulted
in reduced expression of iAR (Fig 3). As shown in Figures 2 and 3, upon
blocking of iAR either by flutamide or by siRNA silencing actin
polymerization was still induced in Caco2 cells treated by testosterone
conjugates, indicating that iAR is not involved in the early actin
reorganization. These findings are in line with previous reports, which
established clearly that neither flutamide treatment nor iAR silencing
inhibited any of the mAR responses tested in prostate cancer cells [3].
Conclusions:
•Activation of membrane Androgen Receptors (mAR) induces rapid actin cytoskeleton reorganization.
•Control experiments using either the anti-androgen flutamide or appropriate siRNAs to silence iAR did not influenced the rapid actin
polymerization.
•These findings imply that actin reorganization is stimulated directly by the membrane androgen binding sites .
•Accordingly, the rapid actin polymerization triggered by testosterone albumin conjugates (TAC) is not correlated with the classical
androgen receptor signaling.
References: [1] S. Gu, N. Papadopoulou, E.M. Gehring, O. Nasir, K. Dimas, S.K. Bhavsar, M. Foller, K. Alevizopoulos, F. Lang, C. Stournaras, (2009) Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce
drastically tumor incidence in vivo, Mol Cancer. 8 114. [2] N. Papadopoulou, I. Charalampopoulos, K. Alevizopoulos, A. Gravanis, C. Stournaras, (2008) Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer
cells, Exp Cell Res. 314 3162-3174. [3] E.A. Papakonstanti, M. Kampa, E. Castanas, C. Stournaras, (2003) A rapid, nongenomic, signaling pathway regulates the actin reorganization induced by activation of membrane testosterone receptors, Mol Endocrinol.
17 870-881. [4] F.M. Vega, A.J. Ridley, (2008) Rho GTAases in cancer cell biology, FEBS Lett. 582 2093-2101.