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: hideakimiyake@hotmail.com

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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