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RESEARCH ARTICLE
Enhanced sensitivity to sorafenib by inhibition of Akt1 expressionin human renal cell carcinoma ACHN cells both in vitroand in vivo
Hiromoto Tei • Hideaki Miyake • Masato Fujisawa
Received: 18 December 2014 / Accepted: 18 February 2015 / Published online: 11 April 2015
� Japan Human Cell Society and Springer Japan 2015
Abstract To investigate whether antitumor activity of
sorafenib, a potential molecular-targeted agent against
RCC is enhanced by silencing Akt1 in a human RCC
ACHN model. We established ACHN in which the ex-
pression vector containing short hairpin RNA targeting
Akt1 was introduced (ACHN/sh-Akt1). Changes in several
phenotypes of ACHN/sh-Akt1 following treatment with
sorafenib were compared with those of ACHN transfected
with control vector alone (ACHN/C) both in vitro and
in vivo. When cultured in the standard medium, there was
no significant difference in the in vitro growth pattern be-
tween ACHN/sh-Akt1 and ACHN/C; however, compared
with ACHN/C, ACHN/sh-Akt1 showed a significantly
higher sensitivity to sorafenib. Furthermore, treatment with
Akt1 inhibitor, A-674563 also resulted in the significantly
enhanced sensitivity of parental ACHN to sorafenib.
Treatment of ACHN/sh-Akt1 with sorafenib, but not that of
ACHN/C, induced marked downregulation of antiapoptotic
proteins, including Bcl-2, Bcl-xL, and c-Myc. In vivo ad-
ministration of sorafenib resulted in the significant growth
inhibition of ACHN/sh-Akt1 tumor compared with that of
ACHN/C tumor, and despite the lack of Ki-67 labeling
index between ACHN/sh-Akt1 and ACHN/C tumors,
apoptotic index in ACHN/sh-Akt1 tumor in mice treated
with sorafenib was significantly greater than that in ACHN/
C tumor. These findings suggest that combined treatment
with Akt1 inhibitor and sorafenib could be a promising
therapeutic approach for patients with advanced RCC.
Keywords Akt1 � Chemosensitivity � Renal cellcarcinoma � Sorafenib
Introduction
Renal cell carcinoma (RCC), the most common malignancy
of the adult kidney, is characterized by the high incidence of
metastatic spread; that is, it has been shown that ap-
proximately 30 % of patients with RCC demonstrate
metastasis at initial diagnosis, and 20–40 % of those with
localized disease who undergo surgical resection with cu-
rative intent subsequently develop metastatic diseases [1]. In
recent years, several types of novel molecular-targeted agent
have been developed based on the precise understanding of
molecular mechanisms mediating the progression of RCC,
and the introduction of these new drugs has resulted in a
dramatic paradigm shift in the therapeutic strategy for
metastatic RCC [2]. Of these, sorafenib, an orally available
multi-targeted receptor tyrosine kinase inhibitor (TKI), has
been shown to have inhibitory effects on tumor cell prolif-
eration as well as angiogenesis in preclinical RCC models
[3]. In a clinical setting as well, the excellent antitumor ac-
tivity of sorafenib against RCC was demonstrated, exhibit-
ing a significantly favorable progression-free survival
compared with a placebo in a phase III randomized trial [4].
However, several limitations of sorafenib as a therapeutic
agent against metastatic RCC have been pointed out, in-
cluding the low proportion of patients achieving a complete
or partial response and the short interval of a durable re-
sponse [5, 6]. Therefore, it would be of interest to develop a
novel therapeutic strategy for metastatic RCC patients to
enhance the efficacy of sorafenib by the combined use of a
drug exerting an antitumor activity through the inactivation
of signaling pathways different from this agent.
H. Tei � H. Miyake (&) � M. Fujisawa
Division of Urology, Kobe University Graduate School of
Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
e-mail: [email protected]
123
Human Cell (2015) 28:114–121
DOI 10.1007/s13577-015-0112-8
Although the detailed regulatory pathways in the pro-
gression of malignant tumors remain poorly elucidated,
there has been accumulated evidence showing an important
role for the activation of Akt in promoting survival as well
as inhibiting apoptotic cell death in a wide variety of
cancer model systems [7]. Furthermore, Akt is known to
consist of three family members: Akt1, Akt2, and Akt3,
with highly conserved domain of serine/threonine kinase
[8]. Of these members, Akt1 is regarded as playing a
dominant role in the regulation of signaling pathway me-
diating the progression of malignant tumors, including
RCC [9–11]. For example, Zeng et al. reported the
pathologic cooperativity in human RCC cells between
PTEN inactivation and loss of von Hippel-Lindau tumor
suppressor which leads to the superactivation of Akt1 [11].
Furthermore, unfavorable disease control by anti-cancer
agents could be explained, at least in part, by intrinsic and/
or acquired resistance in tumors to therapeutic agents, and
there have been various studies showing the direct in-
volvement of persistent activation of Akt signaling path-
ways during treatment with TKIs in the acquisition of a
phenotype resistant to these agents in RCC cells [6, 12–14].
Collectively, these findings suggest that sensitivity of RCC
cells to sorafenib could be further enhanced by the simul-
taneous inactivation of Akt1; therefore, in this study, we
investigated the inhibitory effects of Akt1 expression in
human RCC ACHN cells on changes in their phenotypes
both in vitro and in vivo, focusing on those related to
treatment with sorafenib.
Materials and methods
Tumor cell line
ACHN, derived from human RCC, was purchased from the
American Type Culture Collection (Rockville, MD, USA),
and used within 6 months of resuscitation. Cells were
maintained in RPMI-1640 media (Sigma-Aldrich, St Louis,
MO, USA) at 37 �C in 5 % CO2 and supplemented with
10 % heat-inactivated fetal bovine serum (Invitrogen Life
Technologies Inc., San Diego, CA, USA).
Expression plasmid and transfection to tumor cells
A chemically synthesized oligonucleotide encoding a short
hairpin RNA (shRNA) targeting Akt1 (50-CTACCTG-CACTCGGAGAAGAA-30), including a loop motif, was
inserted downstream to the U6 promoter of the pGene-
ClipTM Neomycin Vector (QIAGEN, Tokyo, Japan).
Similarly, a control plasmid was constructed by random-
izing the sequence of shRNA corresponding to Akt1 gene
(50-GGAATCTCATTCGATGCATAC-30).
Expression vectors were transfected into ACHN cells
using liposome-mediated gene transfer methods [15]. In
brief, either the purified expression plasmid containing
shRNA targeting Akt1 or the control plasmid was added to
ACHN cells after preincubation for 20 min with Lipofec-
tamineTM 2000 and serum-free OPTI-MEM (Invitrogen
Life Technologies Inc.). Drug selection in 1 mg/ml neo-
mycin (Sigma-Aldrich) was started 3 days after transfec-
tion and then colonies were harvested and expanded to cell
lines.
Cell proliferation assay
To compare the in vitro proliferation of ACHN sublines,
5 9 103 cells of each cell line were seeded in each well of
96-well plates and allowed to attach overnight. The number
of cells in each cell line was assessed using a Cell Counting
Kit-8 (Dojindo Molecular Technologies, Kumamoto, Ja-
pan). In addition, the effects of treatment with sorafenib
(LKT Laboratories Inc., St. Paul, MN, USA) either alone or
in combination with 0.1 lMA-674563 (Selleck Chemicals,
Houston, TX, USA) on the proliferation of ACHN sublines
were also examined after 48 h of incubation with various
doses of sorafenib. Each assay was performed in triplicate.
Absorbance was measured at 450 nm using a Benchmark
Plus Microplate reader (Bio-Rad Laboratories, Hercules,
CA, USA).
Western blot analysis
Western blot analysis was performed as described previ-
ously [16]. Samples containing equal amounts of protein
(25 lg) from lysates of the ACHN sublines cultured in
either standard medium or medium containing sorafenib
were subjected to SDS–polyacrylamide gel electrophore-
sis and transferred to a nitrocellulose filter. The filter was
blocked in PBS containing 5 % nonfat milk powder at
4 �C overnight and then incubated for 1 h with antibodies
against total and phosphorylated (p)-Akt1 (Cell Signaling
Technology, Danvers, MA, USA), Bcl-2, Bax, Bcl-xL
(Santa Cruz Biotechnology, Santa Cruz, CA, USA),
myeloid cell leukemia sequence 1 (Mcl1), c-Myc (Cell
Signaling Technology), and b-actin (Santa Cruz
Biotechnology) at dilutions of 1:1000. The filters were
then incubated for 30 min with horseradish peroxide-
conjugated secondary antibodies at dilutions of 1:2000
(Amersham Pharmacia Biotech, Arlington Heights, IL,
USA), and specific proteins were detected using an en-
hanced chemiluminescence western blot analysis system
(Amersham Pharmacia Biotech). The strength of each
signal density was semiquantitatively determined using a
densitometer (Bio-Tek Instruments, Inc., Winooski, VT,
USA).
Enhanced sensitivity to sorafenib by inhibition of Akt1 expression 115
123
Assessment of in vivo tumor growth
Male athymic nude mice (BALB/c-nu/nu males,
6–8 weeks old) were purchased from Clea Japan (Tokyo,
Japan) and housed in a controlled environment at 22 �C on
a 12-h light/12-h dark cycle. Animals were maintained in
accordance with the National Institutes of Health Guide for
the Care and Use of Laboratory Animals. Each ex-
perimental group consisted of 5 mice. The tumor cells of
each ACHN subline were trypsinized, and 5 9 106 cells
were injected subcutaneously with 100 ll of Matrigel
(Becton–Dickinson, Franklin Lakes, NJ, USA). When the
volume of subcutaneous tumor reached approximately
100 mm3, mice were randomly selected for oral adminis-
tration of either sorafenib at a dose of 20 mg/kg or that of
vehicle once daily for 4 weeks. Subcutaneous tumor
growth was measured at least once per week using calipers
and calculated using the formula: length 9 width 9
depth 9 0.5236, as described previously [17].
Histopathological study of in vivo tumor
In vivo subcutaneous tumors were harvested from nude
mice treated with sorafenib or vehicle for 4 weeks ac-
cording to the schedule described above. Immunohisto-
chemical staining of tumor specimens was performed as
previously reported [18]. In brief, sections from
formaldehyde-fixed, paraffin-embedded tissue were de-
paraffinized with xylene and rehydrated in decreasing
concentrations of ethanol. After the blocking of endoge-
nous peroxidase with 3 % hydrogen peroxidase in metha-
nol, sections were stained with antibodies against Ki-67
(Abcam, Cambridge, UK), Bcl-2, Bcl-xL (Santa Cruz
Biotechnology), and c-Myc (Cell Signaling Technology) at
dilutions of 1:200 for 60 min. Sections were subsequently
incubated with biotinylated secondary IgG (Vector
Laboratories, Burlingame, CA, USA) at dilutions of 1:2000
for 30 min. After incubation in avidin–biotin-peroxidase
complex for 30 min, samples were exposed to di-
aminobenzidine tetrahydrochloride solution and counter-
stained with methyl green. In addition, terminal
deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) staining of subcutaneous tumors was performed
using an In Situ Cell Death Detection Kit POD (Roche
Applied Science, Indianapolis, IN, USA), according to the
manufacturer’s instructions.
Statistical analysis
Differences between the two groups were compared using
an unpaired t test and all results were expressed as the
mean ± SD. All statistical calculations were performed
using Statview 5.0 software (Abacus Concepts Inc.,
Berkley, CA, USA), and P\ 0.05 were considered statis-
tically significant.
Results
Akt1 expression in ACHN sublines
ACHN cells were transfected with an expression vector
containing the shRNA targeting Akt1 or the control vector
alone, and after drug selection, a number of independent
stable clones were established. Western blot analysis was
then performed to assess the expression levels of Akt1
protein in parental ACHN (ACHN/P), control vector-
transfected ACHN (ACHN/C), and five picked-up clones
transfected with the vector containing Akt1 shRNA
(ACHN/sh-Akt1#1 to ACHN/sh-Akt1#5). As shown in
Fig. 1, abundant Akt1 expression was observed in ACHN/
P and ACHN/C; however, expression levels of Akt1 in the
5 Akt1 shRNA-transfected clones were markedly inhibited
compared with those in ACHN/P and ACHN/C.
In the following in vitro experiments, similar findings
were obtained from the Akt1 shRNA-transfected cell lines
(ACHN/sh-Akt1#1 to ACHN/sh-Akt1#5) or the control
cell lines (ACHN/P and ACHN/C); therefore, only the data
for ACHN/sh-Akt1#2 and ACHN/C were subsequently
presented.
In vitro growth of ACHN sublines
To assess the effect of decreased Akt1 expression on the
growth ofACHNcells, the in vitro growth patterns of ACHN
sublines were compared. No significant difference in the
growth patterns between ACHN/C and ACHN/sh-Akt1#2
was noted, when cultured in the standard medium (Fig. 2a).
Akt1
β-actin
AC
HN
/P
AC
HN
/C
AC
HN
/sh-
Akt
1#1
AC
HN
/sh-
Akt
1#2
AC
HN
/sh-
Akt
1#3
AC
HN
/sh-
Akt
1#4
AC
HN
/sh-
Akt
1#5
Fig. 1 Expression levels of Akt1 in ACHN sublines. (ACHN/P
parental cell line, ACHN/C control vector-only transfected cell line,
ACHN/sh-AKt1#1 to #5 Akt1 short hairpin RNA-transfected cell
lines). Protein was extracted from each cell line, and Western blotting
was performed to analyze the expression levels of Akt1 and b-actin in
ACHN sublines
116 H. Tei et al.
123
To determine whether the inhibition of Akt1 expression
enhances the sensitivity of ACHN cells to sorafenib,
ACHN sublines were treated with various concentrations of
sorafenib. As shown in Fig. 2b, ACHN/sh-Akt1#2 was
more sensitive to sorafenib than ACHN/C, that is, the IC50
of sorafenib in ACHN/sh-Akt1#2 was reduced by ap-
proximately 90 % compared with that in ACHN/C. Fur-
thermore, p-Akt1 expression in ACHN/sh-Akt1#2 was
significantly lower than that in ACHN/C, when cultured in
both standard medium alone and that with sorafenib at a
concentration of 1.0 lM (Fig. 2c).
To confirm the impact of Akt1 inhibition on the en-
hanced sensitivity of ACHN cells to sorafenib, growth in-
hibitory effects of various doses of sorafenib either alone or
in combination with Akt1 inhibitor, A-674563, on ACHN/
P were assessed. As shown in Fig. 2d, additional treatment
with A-674563 resulted in the significant increase in the
sensitivity of ACHN/P to sorafenib.
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10 100
ACHN/C
ACHN/sh-Akt1#2
Num
ber o
f cel
ls (A
rbitr
ary
units
)
Days in culture
a b
Concentration of sorafenib (μM)
Num
ber o
f cel
ls (A
rbitr
ary
units
)
*
** ** **
0
1
2
3
4
5
6
7
1 2 3 4 5
ACHN/CACHN/sh-Akt1#2
cACHN/sh-Akt1#2
Sorafenib (μM)
ACHN/C
(–) (–) 1.0 5.0 1.0 5.0
p-Akt1
d
Akt1
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10 100
Without A-674563
With A-674563Num
ber o
f cel
ls (A
rbitr
ary
units
)
Concentration of sorafenib (μM)
*
*
**
Fig. 2 a In vitro cell growth of ACHN sublines. In vitro proliferation
of ACHN/C and ACHN/sh-Akt1#2 were measured daily by counting
the number of cells in each cell line in triplicate. Bars, SD. b Effect of
treatment with sorafenib on in vitro cell growth of ACHN sublines.
ACHN/C and ACHN/sh-Akt1 #2 were treated with the indicated
doses of sorafenib. After 48 h of incubation, cell growth was
determined in triplicate by counting in three independent ex-
periments. c Changes in expression patterns of phosphorylated (p)-
and total Akt1 in ACHN sublines following treatment with sorafenib.
Expression levels of p-Akt1 and Akt1 in ACHN sublines before and
after treatment with sorafenib at concentration of 1.0 and 5.0 lMwere analyzed by Western blotting. d Effect of treatment with
sorafenib either alone or in combination with Akt1 inhibitor,
A-674563, on in vitro cell growth of ACHN/P. After 48 h of
incubation, cell growth was determined in triplicate by counting in
three independent experiments. ** and *, differ from ACHN/P
without A-674563 (P\ 0.01 and P\ 0.05, respectively)
Enhanced sensitivity to sorafenib by inhibition of Akt1 expression 117
123
Expression of key molecules associated with apoptosis
in ACHN sublines
Changes in the expression patterns of apoptosis-related
molecules in ACHN sublines before and after the admin-
istration of sorafenib are presented in Fig. 3. There were no
significant differences in the expression levels of Bax and
Mcl-1 between ACHN sublines cultured in media with and
without sorafenib. When cultured in the medium contain-
ing sorafenib at a concentration of 1.0 lM, expression
levels of Bcl-2 and Bcl-xL in ACHN/C, but not those in
ACHN/sh-Akt1#2, were markedly upregulated; however,
there was no significant difference in the expression of Bcl-
2 and Bcl-xL between ACHN sublines after treatment with
sorafenib at a concentration of 5.0 lM. In addition, c-Myc
expression in ACHN/sh-Akt1#2 was significantly lower
than that in ACHN/C before treatment with sorafenib.
In vivo growth of ACHN sublines
To compare in vivo growth of ACHN sublines with and
without treatment with sorafenib, 5 9 106 cells of each cell
line were subcutaneously injected into nude mice, which
were then randomly applied to treatment with either so-
rafenib or vehicle. As shown in Fig. 4a, there was no sig-
nificant difference in the in vivo growth pattern between
ACHN sublines treated with vehicle. However, despite the
definitive growth suppression of both ACHN sublines by
the administration of sorafenib, significantly marked
growth inhibitory effect of sorafenib treatment on ACHN/
sh-Akt1#2 tumors was observed compared with that on
ACHN/sh-Akt1#2 tumors, that is, the size of ACHN/sh-
Akt1#2 tumors was approximately half as much as that of
ACHN/C tumors.
To confirm the in vitro findings, in vivo expression
levels of Bcl-2, Bcl-xL, and c-Myc in ACHN sublines were
evaluated using immunohistochemical staining. As shown
in Fig. 4b, the expression levels of Bcl-2, Bcl-xL, and
c-Myc in ACHN/C tumors treated with sorafenib were
markedly upregulated compared with those in ACHN/sh-
Akt1#2 tumors. We then compared the sorafenib-induced
changes in cell proliferative as well as apoptotic features in
ACHN sublines in vivo. No significant difference in the
expression pattern of Ki-67 was noted between ACHN/C
and ACHN/sh-Akt1#2 tumors irrespective of treatment
with sorafenib. In sorafenib-treated mice, however,
TUNEL assay showed a significantly greater proportion of
cells undergoing apoptosis in ACHN/sh-Akt1#2 tumors
than that in ACHN/C tumors (Fig. 4b).
Discussion
Despite recent introduction of several newly approved
agents into the clinical practice, it remains difficult to
achieve a satisfactory disease control in patients with
metastatic RCC [19–21]; thus, it would be necessary to
develop a novel therapeutic strategy for further improving
the survival of patients with this disease. It could be an
attractive approach for this objective to enhance the ac-
tivity of an existing agent by combining a new drug with a
unique mechanism of action. Sorafenib, an orally available
multiple TKI, has been shown to have comparatively low
antitumor activity against RCC as a single agent [22];
however, to date, there have been several studies demon-
strating the improved therapeutic potential of this agent by
an additional pharmacological modulation [18, 23, 24]. For
example, we previously reported that a combined use with
OGX-011, antisense oligonucleotide targeting clusterin,
enhanced the cytotoxic effect of sorafenib on RCC cells
through the marked down-regulation of p-Akt [18]. Con-
sidering these findings, we analyzed the significance of
silencing Akt1, the most potential member of the Akt
family, in the enhancement of sensitivity to sorafenib in
human RCC ACHN model both in vitro and in vivo.
In this study, when cultured in the standard medium,
despite markedly higher expression of p-Akt in ACHN/C
than that in ACHN/sh-Akt1#2, no significant difference in
the growth between these sublines was noted. However,
conflicting findings concerning whether it is sufficient to
inhibit the growth of cancer cells by decreasing the ex-
pression of Akt1 alone have been reported [25, 26], which
might be explained by several reasons, such as the degree
of Akt1 inhibition and differences in the characteristics of
ACHN/sh-Akt1#2
Sorafenib (μM)
ACHN/C
(-) (-) 1.0 5.0 1.0 5.0
Bax
Bcl-xL
Mcl-1
c-Myc
β-actin
Bcl-2
Fig. 3 Changes in expression patterns of key molecules involved in
apoptosis in ACHN sublines following treatment with sorafenib.
Expression levels of Bcl-2, Bcl-xL, Bax, Mcl-1, c-Myc and b-actin in
ACHN sublines before and after treatment with sorafenib at
concentrations of 1.0 and 5.0 lM were analyzed by Western blotting
118 H. Tei et al.
123
0
100
200
300
400
500
600
0 4 9 13 19 26 30
ACHN/C
ACHN/sh-Akt1#2
ACHN/C
ACHN/sh-Akt1#2
Tum
ourv
olum
e (m
m3 )
Day after treatment
Vehicle
Sorafenib
a
*
***
Sorafenib (-) Sorafenib (+)
Bcl-2
Bcl-xL
b ACHN/c ACHN/sh-Akt1#2
Sorafenib (+)Sorafenib (-)
TUNEL
Ki-67
c-Myc
Enhanced sensitivity to sorafenib by inhibition of Akt1 expression 119
123
cell lines among these studies. We subsequently revealed
that the administration of sorafenib resulted in the sig-
nificantly marked growth inhibition in ACHN/sh-Akt1#2
compared with that in ACHN/C. Furthermore, phospho-
rylated status of Akt1 in both ACHN sublines was shown to
be inversely proportional to the growth inhibitory effects
induced by sorafenib. To date, there have been several
studies illustrating the close relation between the Akt1
expression in cancer cells and their susceptibilities to anti-
cancer drugs [27–29]. For example, Yanagihara et al. [29]
reported that downregulation of Akt1 expression using ri-
bozymes targeting Akt1 sensitized human cancer cells to
typical chemotherapeutic agents. Collectively, these find-
ings strongly suggest that cytotoxic activity of anti-cancer
agents, such as sorafenib, could be synergistically en-
hanced by the downregulation of Akt1 in cancer cells.
It is of interest to investigate the molecular mechanism
mediating the enhanced cytotoxicity of sorafenib on ACHN/
sh-Akt1#2. In this study, we analyzed the changes in the ex-
pression patterns of major molecules associated with signal
transduction and apoptosis in ACHN sublines after treatment
with sorafenib. Although therewere no significant differences
in the activated status of signaling pathways between ACHN
sublines following sorafenib treatment (data not shown), the
expression levels of antiapoptotic proteins, including Bcl-2,
Bcl-xL, and c-Myc, in ACHN/sh-Akt1#2 appeared to be
markedly downregulated compared with those in ACHN/C.
There have been several previous studies supporting the
findings on the involvement of apoptosis-related proteins in
the growth inhibition of cancer cells by either the inhibition of
Akt1 expression alone or in combination with cytotoxic
agents [30–32]. For example, Yang et al. [32] found that
DNAzyme targeting Akt1 decreased in the proliferation of
nasopharyngeal carcinoma cells accompanying the induction
of suppressed Bcl-2 as well as increased Bax expression.
Another point of interest is to examine the growth pat-
terns of ACHN sublines in vitro reflect those in vivo, since
changes in a susceptibility of cancer cells to a targeted agent
may modulate gene expression profile, resulting in the
modifications in various accompanying molecular events,
including apoptosis, signal transduction, and angiogenesis.
In this study, the synergistic inhibitory effect of Akt1
downregulation and sorafenib treatment on in vivo ACHN
tumor growth was confirmed, and intensive induction of
apoptotic cell death was observed in ACHN/sh-Akt1#2 tu-
mors in mice receiving sorafenib. Furthermore, similar to
in vitro study, the suppression of Bcl-2, Bcl-xL, and c-Myc
was also documented in ACHN/sh-Akt1#2 tumors in mice
treated with sorafenib. Accordingly, we believe that this
therapeutic animal model for RCC by Akt1 inhibition in
combination with sorafenib could be applied to investigate
the mechanism underlying the cytotoxicity of this therapy
in vivo. In fact, increased phosphorylation of Akt1 in
ACHN/C after treatment with moderate dose of sorafenib
in vitro, which acts as a proapoptotic trigger, represents an
adaptive mechanism mediating cell survival; therefore, ac-
tivation of Akt1 could be responsible for mediating the ac-
quired resistance to sorafenib in RCC.
Here, we would like to emphasize several limitations of
this study. Initially, all outcomes presented in this study
were derived from the data using a single RCC cell line,
ACHN, although we have already achieved findings with a
human prostate cancer cell line, PC3, similar to those
shown in this study (data not shown). Second, the
mechanism related to the findings observed in this study
was investigated focusing on the apoptosis; however, other
molecular events, such as angiogenesis and epithelial
mesenchymal transition, could be more preferentially in-
volved in these findings. Third, it should be investigated
whether similar findings shown in this study could be
achieved by sunitinib, another potential TKI, which is
considered as first-line agent for metastatic RCC. Although
we have already confirmed higher sensitivity to sunitinib in
ACHN/sh-Akt1#2 than that in ACHN/C, the difference in
the sensitivity to sunitinib between ACHN sublines was not
marked compared with that to sorafenib (data not shown).
Finally, although Akt1 is regarded as the most important
member of the Akt family as a mediator of function
regulating cancer progression, it is required to examine the
roles of the remaining members to draw a more precise
conclusion in the issues analyzed in this study.
In conclusion, suppression of Akt1 expression in human
RCC ACHN cells using shRNA technology significantly
enhanced the sensitivity of these cells to sorafenib both
in vitro and in vivo through the regulation of molecules
associated with apoptosis. Therefore, combined treatment
with Akt1 inhibitor and sorafenib could be a promising
therapeutic approach for patients with advanced RCC.
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ACHN sublines. Twenty nude mice were subcutaneously given
5 9 106 cells of each ACHN subline, then randomly selected for
treatment with either 20 mg/kg sorafenib or vehicle five times per
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