ORIGINAL ARTICLE

Curcumin targets the AKT–mTOR pathway for uterineleiomyosarcoma tumor growth suppression

Tze Fang Wong • Takashi Takeda •

Bin Li • Kenji Tsuiji • Akiko Kondo •

Mari Tadakawa • Satoru Nagase • Nobuo Yaegashi

Received: 1 October 2012 / Accepted: 10 April 2013 / Published online: 11 May 2013

� Japan Society of Clinical Oncology 2013

Abstract

Background Uterine leiomyosarcomas generally do not

respond well to standard chemotherapy. We previously

demonstrated that curcumin, the active ingredient derived

from the herb Curcuma longa, inhibits uterine leiomyo-

sarcoma cells in vitro via the inhibition of the AKT–

mammalian target of rapamycin (mTOR) pathway. As a

preclinical investigation, we performed an in vivo study

using female nude mice to confirm the therapeutic potential

of curcumin against uterine leiomyosarcoma.

Methods Human leiomyosarcoma cells, SK-UT-1, were

inoculated in female nude mice to establish subcutaneous

tumors. Either vehicle control or 250 mg/kg curcumin was

administered intraperitoneally every day for 14 consecutive

days, and the mice were then killed. The tumors were measured

every 2–3 days. The tumors were processed for immunohis-

tochemical analyses to detect total AKT, phosphorylated AKT,

total mTOR, phosphorylated mTOR, and phosphorylated S6.

To detect apoptosis, the tumors were stained for cleaved PARP

and TUNEL. Ki-67 immunohistochemistry was performed to

determine cell viability of the tumors.

Results Compared with the control, curcumin reduced

uterine leiomyosarcoma tumor volume and mass signifi-

cantly with a concordant decrease in mTOR and S6

phosphorylation. However, AKT phosphorylation was not

significantly altered. Cleaved PARP and TUNEL staining

increased significantly with curcumin administration,

indicating the induction of apoptosis. There was no dif-

ference in Ki-67 staining between the two groups.

Conclusion Curcumin inhibited uterine leiomyosarcoma

tumor growth in vivo by targeting the AKT–mTOR path-

way for inhibition.

Keywords Curcumin � Uterine leiomyosarcoma �AKT–mTOR pathway � Apoptosis � In vivo

Introduction

Uterine leiomyosarcoma is a rare malignant tumor that com-

prises 1 % of all malignancies arising from the uterus.

Because of its generally low response rate to standard adjuvant

chemotherapy, high propensity for hematological metastases,

and high frequency of recurrence, uterine leiomyosarcoma is

especially difficult to treat. Currently, adjuvant treatment with

doxorubicin shows the best efficacy to toxicity ratio profile for

uterine leiomyosarcomas and has been the first-line therapy

for the past 20 years [1]. Various other cytotoxic chemicals

such as gemcitabine, docetaxel, etc. have been tested either

alone or in combination, but no promising alternative to

doxorubicin monotherapy has been discovered so far. Clinical

trials to test molecular targeted approaches are under way.

Hormone-targeted therapy using antiprogestin [2] or aroma-

tase inhibitors [3], mammalian target of rapamycin (mTOR)

inhibition using rapamycin analogues [4], and antiangiogen-

esis using either monoclonal antibodies or target-specific

small molecules [5] are some of the approaches being evalu-

ated at various stages of clinical trials.

The constitutive activation of the mTOR pathway is one

of the most investigated mechanisms of tumorigenesis of

T. F. Wong � T. Takeda � B. Li � K. Tsuiji � A. Kondo �M. Tadakawa � S. Nagase � N. Yaegashi

Department of Obstetrics and Gynecology, Tohoku University

Graduate School of Medicine, Sendai, Miyagi, Japan

Present Address:

T. Takeda (&)

Division of Women’s Health, Research Institute of Traditional

Asian Medicine, Kinki University School of Medicine,

377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan

e-mail: take@med.kindai.ac.jp

123

Int J Clin Oncol (2014) 19:354–363

DOI 10.1007/s10147-013-0563-4

leiomyosarcoma. Mice harboring smooth muscle-specific

knockout of the PTEN (phosphatase and tensin homologue

deleted from chromosome 10) gene died prematurely of

leiomyosarcoma; their survival was prolonged by treatment

with everolimus, a rapamycin analogue [6]. Incidentally,

59 % of uterine and extrauterine leiomyosarcoma cases

reported in one study had deletions of the chromosomal

region 10q that contains the PTEN gene [7]. Taken toge-

ther, these findings provide the evidence backing the tar-

geted approach against the mTOR pathway.

Our previous investigation explored the possibility of

using curcumin, an active ingredient derived from the

Indian spice, Curcuma longa, as a therapeutic candidate

against uterine leiomyosarcoma. Curcumin at a concen-

tration exceeding 25 lM in vitro was able to reduce uterine

leiomyosarcoma cell growth by more than 50 % and

induce apoptosis [8]. Building on the discovery that cur-

cumin inhibits mTOR in other tumor cells such as rhab-

domyosarcoma [9] and prostate cancer [10], we previously

demonstrated that curcumin was able to suppress mTOR

activity by downregulating the phosphorylation of mTOR

(Ser2448) and its downstream effectors, p70S6 (Thr389)

and S6 (Ser235/236), in uterine leiomyosarcoma cells.

The potential of curcumin as an anticancer agent has been

explored in numerous studies. Despite its widely touted

efficacy against many cancer types regardless of their tissue

origins, curcumin has been limited by its poor bioavailability

and pharmacodynamics [11]. The most preferred mode of

administration for curcumin was the oral route, which has

been investigated in mouse models of ovarian cancer [12],

breast cancer [13], and prostate cancer [14]. One compre-

hensive study on the bioavailability of curcumin that com-

pared the oral route (p.o.) with intraperitoneal (i.p.)

administration suggests that i.p. administration is 100 times

(maximum plasma curcumin concentration, 2.25 lg/ml, at

100 mg/kg i.p.) more efficient than oral administration

(maximum plasma curcumin concentration, 0.22 lg/ml, at

1000 mg/kg p.o.) [15]. Thus, we have chosen to test curcu-

min’s action against a uterine leiomyosarcoma mouse model

in vivo using intraperitoneal curcumin administration. By

investigating curcumin’s tumor suppressive effect and

its effect on mTOR activity in vivo, we hope that our cur-

rent study will provide the necessary rationale for using

curcumin against uterine leiomyosarcoma in future clinical

investigations.

Materials and methods

Chemicals

Curcumin (from Curcuma longa, turmeric) and corn oil

were purchased from Wako Pure Chemical Industries

(Osaka, Japan). Curcumin was dissolved in dimethyl sulf-

oxide (DMSO) (Sigma–Aldrich, St. Louis, MO, USA) at

1 M concentration as a stock solution that was stored at

-20 �C. For intraperitoneal administration of curcumin,

curcumin was dissolved in 20 % DMSO and suspended in

corn oil at 250 mg/kg/50 ll per animal.

Cell culture

The uterine LMS cell line, SK-UT-1 was purchased from

American Type Culture Collection (ATCC, Manassas, VA,

USA). The cells were revived in Eagle’s minimum essen-

tial medium (Gibco, Invitrogen, Carlsbad, CA, USA)

supplemented with 10 % fetal bovine serum (FBS) and

maintained at 37 �C in a humidified 5 % CO2 atmosphere.

Recipient animal and tumor model

A uterine LMS tumor model was established in BALB/cAJC

female nude mice using SK-UT-1 tumor cells. Briefly, female

mice 5–6 weeks old were purchased from CLEA Japan and

housed in a specific-pathogen-free animal facility at the

Center for Laboratory Animal Research, Tohoku University

Graduate School of Medicine, with the approval from the

University’s committee on laboratory animal research. The

research was conducted in accordance with the regulations

laid out by the Ministry of Education, Culture, Sports, Science

and Technology, the Ministry of Health, Labour and Welfare,

and the Ministry of the Environment, Japan.

Before subcutaneous implantation of SK-UT-1 tumor

cells into nude mice, the cells were grown in monolayer

culture in medium supplemented with 20 % FBS: 107 SK-

UT-1 cells were suspended in 0.05 ml medium mixed with

0.05 ml Matrigel (Becton, Dickinson, Franklin Lakes, NJ,

USA), and then injected subcutaneously into both flanks of

mice. Subcutaneous tumor nodules appeared 7 days po-

stimplantation. Daily intraperitoneal curcumin administra-

tion (250 mg/kg/50 ll) was started on the 8th day

postimplantation. Control mice received intraperitoneal

injections of 20 % DMSO suspended in corn oil (total

volume, 50 ll per animal) as curcumin was dissolved in the

same ratio of DMSO to corn oil. The day of the start of

treatment was counted as day 1. The tumor volume was

measured every 1–3 days and was calculated as previously

described [16]. The length, width, and depth of the tumors

were measured and the volume calculated using the fol-

lowing formula: volume = R1 9 R2 9 R3 9 0.52. The

mice were killed after 14 days of treatment.

Immunohistochemistry

The subcutaneous tumors harvested were formalin fixed

and paraffin embedded. Immunohistochemical analyses

Int J Clin Oncol (2014) 19:354–363 355

123

were performed using a streptavidin–biotin amplification

method using the Histofine Kit (Nichirei, Kyoto, Japan).

Briefly, paraffin-embedded tumors were sliced, mounted on

slides, and deparaffinized for immunohistochemistry. The

slides were autoclaved at 121 �C for 5 min in citrate buffer

(pH 6.0) for antigen retrieval. For detection of the AKT–

mTOR pathway activity, antibodies detecting phosphory-

lated AKT (Thr308; P-AKT), total AKT, phosphorylated

mTOR (Ser2448; P-mTOR), total mTOR, and phosphory-

lated S6 (Ser235/236; P-S6) were purchased from Cell

Signaling Technology (Beverly, MA, USA). The staining

as defined by H scores was calculated as the product of

intensity (0–3) and percentage of area positively stained

(score range: 0–300). At least three independent micro-

scopic fields were evaluated from each sample. To check

the viability of tumor cells, antibody against Ki-67 was

purchased from Dako (Carpinteria, CA, USA). Ki-67

staining was calculated as the percentage of nuclei posi-

tively stained. For the detection of apoptosis, antibodies

detecting cleaved PARP (Cell Signaling Technology) and

TdT-mediated dUTP nick end labeling (TUNEL) staining

were used. TUNEL staining was performed using an

In Situ Cell Death Detection Kit, POD (Roche Diagnostics,

Basel, Switzerland) according to the manufacturer’s

instructions. Both cleaved PARP and TUNEL staining

were scored as the number of nuclei positively stained in

2,000 cells counted, referred to as apoptotic index (AI).

This calculation was based on a method previously

described [17]. The slides were observed and the images

taken using a Nikon Coolscope Digital Microscope (Nikon,

Tokyo, Japan).

Statistical analysis

Each experimental group experiment consisted of five mice

(n = 5). Student’s t test was used to evaluate statistical

significance. The statistical software JMP (SAS, Cary, NC,

USA) was used for analyses and graphical representations.

Differences were considered statistically significant at

p \ 0.05.

Results

Curcumin inhibits uterine leiomyosarcoma tumor

growth in vivo

Intraperitoneal administration of 250 mg/kg curcumin

dissolved in DMSO and corn oil significantly (p \ 0.05)

inhibited uterine leiomyosarcoma tumor growth compared

to tumors in control animals from day 9 (Fig. 1a). Tumors

in animals treated with curcumin continued to grow, albeit

at a slower pace, until the animals were killed at day 14,

and the size of tumors remained significantly smaller than

in the control group (curcumin, 138.0 ± 91.4 mm3, vs.

control, 291.1 ± 133.8 mm3; p \ 0.01) (Fig. 1b). Tumors

excised from the animals were weighed (results shown

in Fig. 1c). Tumors treated with curcumin had significantly

smaller mass compared with the control group (curcu-

min, 314.7 ± 163.0 mg, vs. control, 489.3 ± 170.1 mg;

p \ 0.05).

Curcumin downregulates mTOR (Ser2448) and S6

(Ser235/236) phosphorylation, but not AKT

phosphorylation (Thr308) in vivo

Our previous in vitro data suggest that curcumin inhibits

uterine leiomyosarcoma cell growth by inhibiting the

activity of mTOR. We detected the phosphorylation of

AKT, mTOR, and S6 in the excised tumors using immu-

nohistochemistry of paraffin-embedded tissue slides. Fig-

ure 2a shows representative images of tumors from both

control and curcumin-treated groups. From gross observa-

tion, curcumin seemed to lower the intensity and the area of

positive staining for mTOR and S6, whereas AKT phos-

phorylation seemed to increase slightly. Figure 2b shows

box graphs representing the results of quantification of

P-AKT, P-mTOR, and P-S6 staining. As shown, curcumin

treatment significantly downregulated mTOR (H score:

curcumin, 10.2 ± 6.4, vs. control, 18.2 ± 8.9; p \ 0.05)

and S6 phosphorylation (H score: curcumin, 12.5 ± 16.3,

vs. control, 48.5 ± 39.0; p \ 0.05) in the tumors.

The effect of curcumin on total AKT and total mTOR

expression in vivo was also investigated. Total mTOR

expression was also reduced with curcumin treatment

although the effect was not statistically significant

(H score: curcumin, 125 ± 38.1, vs. control, 97.5 ± 29.0;

p = 0.0859). Both AKT phosphorylation (H score: curcu-

min, 45.5 ± 22.0, vs. control, 27.0 ± 17.5; p = 0.0523)

and total AKT expression (H score: curcumin,

112.0 ± 49.4, vs. control, 91.5 ± 26.5; p = 0.2625)

increased slightly as a result of treatment with curcumin

but the results were not statistically significant.

Curcumin induces apoptosis in uterine leiomyosarcoma

subcutaneous xenograft

Our previous in vitro data demonstrated that curcumin

induces apoptosis in uterine leiomyosarcoma cells. We

detected cleaved PARP and fragmented DNA (TUNEL

staining) in the excised tumors using immunohistochem-

istry of paraffin-embedded tissue slides. Figure 3a shows

representative images of tumors from both control and

curcumin-treated groups. From gross observation, curcu-

min seemed to increase the number of apoptotic cells in the

tumors. Figure 3b,c shows box graphs representing the

356 Int J Clin Oncol (2014) 19:354–363

123

quantification of positive cleaved PARP and TUNEL

staining. As shown, curcumin treatment significantly

increased PARP cleavage (AI: curcumin, 9.2 ± 3.3, vs.

control, 5.0 ± 2.8; p \ 0.01) and DNA fragmentation (AI:

curcumin, 8.3 ± 4.0, vs. control, 4.6 ± 2.6; p \ 0.05) in

the tumors.

Curcumin does not affect Ki-67 expression in viable

cells

Ki-67 is frequently used to evaluate the proliferative abil-

ities of cells within a tissue. Figure 4a shows representative

images of tumors treated with either curcumin or control.

No obvious difference was observed between the two

groups. Figure 4b is a graph showing the quantified stain-

ing percentage using anti-Ki-67 antibody. Curcumin did

not significantly alter Ki-67 expression in the tumors,

although the average staining was slightly reduced, to

39.7 ± 8.4 % from 44.5 ± 7.6 % of the control group

(p = 0.202).

Treatment with curcumin is well tolerated in vivo

Standard chemotherapy often causes side effects such as

anorexia, nausea, vomiting, diarrhea, and alopecia that may

affect the overall well-being of the individual treated. To

Fig. 1 Uterine leiomyosarcoma SK-UT-1 subcutaneous tumor

growth in female nude mice, n = 5 per group. a Tumor growth in

control (squares) versus 250 mg/kg curcumin (triangles). The tumors

were measured with a caliper. Graphical representation of tumor

volume on the 14th day of treatment (b) and tumor weight on the 14th

day of treatment (c). *p \ 0.05; **p\ 0.01; curcumin treatment

compared with control, validated using Student’s t test

Int J Clin Oncol (2014) 19:354–363 357

123

assess the toxicity of curcumin in vivo, the gross appear-

ance of the mice treated was observed at regular intervals

and their weight measured at least thrice weekly. Figure 5

shows the average weight of the mice in the two treatment

groups. As shown, no significant weight loss was observed

in the group treated with intraperitoneal 250 mg/kg cur-

cumin. According to our observation, curcumin did not

significantly alter the gross appearance and behavior of the

mice.

Discussion

Many in vitro and in vivo studies have documented the

anticancer effect of curcumin in malignancies originating

from the ovary [12], breast [13], prostate [14], brain [18],

lymph [19], and lung [20], and the list goes on. A diet

mixed with 2 % w/w curcumin fed to a breast cancer

xenograft mouse model (that used a highly metastatic

cell line, MDA-MB-435) was able to suppress lung

Fig. 2 Curcumin effect on

AKT–mTOR pathway in vivo.

a Representative micrographs of

the tumors. 95. Graphical

representation of AKT

phosphorylation (b), total AKT

expression (c), mTOR

phosphorylation (d), total

mTOR expression (e), and S6

phosphorylation in vivo

(f) (H score). *p \ 0.05; N.S

(not significant); curcumin

treatment compared with

control, validated using

Student’s t test

358 Int J Clin Oncol (2014) 19:354–363

123

metastasis. The study did not compare the tumor size

between treatment groups, but was able to demonstrate

that curcumin significantly downregulated Ki-67 expres-

sion in the tumors [13]. An in vivo ovarian cancer model

showed that daily oral curcumin at 500 mg/kg dosage

could reduce tumor growth by as much as 50 %,

although only marginal statistical significance was

achieved [12]. In the prostate cancer mouse model, 5 mg/

day curcumin was fed to mice harboring prostate cancer

cells PC-3 xenografts and tumor growth was monitored

for at least 30 days [14]. In this study, curcumin was

able to suppress tumor growth by almost 50 % compared

with the control, although the statistical significance of

this finding was not mentioned.

Daily intratumoral injection of 100 mg/kg curcumin in an

in vivo glioma mouse model significantly reduced the size of

the subcutaneous tumor xenografts compared with the con-

trol group [18]. This study demonstrated that curcumin

inhibits the AKT–mTOR pathway in glioma cells in vitro,

but the tumors excised were only analyzed for LC-3 (a

marker for autophagy) expression. In a melanoma mouse

model, curcumin was administered intraperitoneally, similar

Fig. 2 continued

Int J Clin Oncol (2014) 19:354–363 359

123

to our study. The dosage used was 25 mg/kg daily, and no

therapeutic effect was observed when curcumin was

administered alone. However, in mice inoculated with anti-

bodies directed against the melanoma cells, curcumin was

able to prolong their survival compared with the untreated

controls [21].

In vivo studies investigating curcumin’s effect in mouse

xenograft models so far could not consistently demonstrate

curcumin’s efficacy. Our study showed that 250 mg/kg

curcumin administered intraperitoneally was able to slow

uterine leiomyosarcoma tumor growth, and the effect was

statistically significant. Although other studies have dem-

onstrated curcumin’s inhibitory effect on various other

pathways, we are the first to show that intraperitoneal

curcumin could inhibit mTOR, S6 phosphorylation, indi-

cating that curcumin is indeed effective against the AKT–

mTOR pathway in vivo. More notably, we have quantified

the effect of curcumin on the tumor tissues and analyzed

the results for statistical significance.

We attribute our favorable results to two factors: first,

the intraperitoneal route of administration, which has

shown higher bioavailability for curcumin in vivo [15]; and

second, a sufficiently high dose, 250 versus 25 mg/kg in

the melanoma mouse model [21]. Assuming that curcumin

bioavailability in nude mice is similar with the study pre-

viously described [15], 250 mg/kg curcumin will yield a

maximum plasma concentration of 5.63 lg/ml or 15.3 lM.

This concentration should be sufficiently high to cause

cytotoxicity to SK-UT-1 cells, as predicted by our previous

in vitro study [8].

We noticed that intraperitoneal administration of

250 mg/kg curcumin could not reduce AKT phosphory-

lation. In contrast, P-AKT staining increased slightly in

the tumor cells in the curcumin-treated group. Our

Fig. 3 Curcumin effect on apoptosis in vivo. a Representative

micrographs of the tumors. 920. Graphical representation of PARP

cleavage (b) and TUNEL staining in vivo (c). AI apoptotic index,

number of darkly stained nuclei in 2,000 cells calculated. *p \ 0.05;

**p \ 0.01; curcumin treatment compared with control, validated

using Student’s t test

360 Int J Clin Oncol (2014) 19:354–363

123

previous investigation on uterine leiomyosarcoma cells

in vitro suggested that low curcumin concentration

could not inhibit AKT phosphorylation [22]. A higher

concentration or enhanced bioavailability using an adju-

vant such as epigallocatechin-3-gallate (EGCG) was nee-

ded to effectively inhibit AKT activity. Perhaps this was

the reason why intraperitoneal 250 mg/kg curcumin daily

could not entirely eliminate uterine leiomyosarcoma in our

mouse model. Sustained AKT phosphorylation as a result

of low plasma curcumin concentration probably enabled

the survival of the tumor cells, as AKT not only reacti-

vates mTOR, but also suppresses apoptosis [23]. It has

been shown that specific inhibition of mTOR by rapa-

mycin causes a negative feedback reactivation of AKT [8,

9]. Although low curcumin concentrations can inhibit

mTOR, a higher concentration is required to inhibit AKT

as well.

Moreover, curcumin has varying affinity for different

kinases [24]. The inhibitory effect of curcumin was

compared between protein kinase A (PkA), protein kinase

C (PkC), protamine kinase (cPK), phosphorylase kinase

(PhK), autophosphorylation-activated protein kinase

(AK), and pp60c-src tyrosine kinase. At about 0.1 mM

(100 lM) curcumin, PhK, pp60c-src, PkC, PkA, AK, and

cPK were inhibited by 98 , 40 , 15 , 10 , l , and 0.5 %,

respectively. Therefore, it is considered reasonable that

AKT requires a higher concentration of curcumin to be

inhibited because different proteins have different affini-

ties for curcumin.

Fig. 4 Curcumin effect on

tumor cell viability in vivo.

a Representative micrographs of

the tumors. 920. Graphical

representation of Ki-67 staining

in vivo (b). N.S, statistically not

significant; curcumin treatment

compared with control,

validated using Student’s t test

Fig. 5 Posttreatment body weight of nude mice (g). Black bars,

control; white bars, curcumin 250 mg/kg

Int J Clin Oncol (2014) 19:354–363 361

123

Excluding clinical trials investigating chemoprevention

using curcumin, phase I or II clinical trials using curcumin

so far were only conducted on pancreatic cancer cases [25,

26]. Curcumin was administered orally in these studies.

Although curcumin was generally well tolerated clinically

and produced very few adverse effects, the plasma con-

centration of curcumin ranged from 29 to 412 ng/ml [27],

which translates to a maximum concentration of

7.87–111.84 nM. This concentration is well below the

cytotoxic dose against cancer cells as predicted by in vitro

studies [28]. As a result, oral curcumin in those clinical

trials failed to significantly improve the survival of the

patients, and the average response rates were about 3 %.

Experimenting with different modes of administration

has certainly reached its limitation in improving curcumin

bioavailability. As demonstrated by our previous study [22]

and another group that combined piperine with curcumin

[29], adding an adjuvant may improve the cellular uptake

and absorption of curcumin in the digestive tract. Another

approach is to prepare curcumin as encapsulated nanopar-

ticles [30]. Such an approach has proven to improve oral

bioavailability of curcumin by as much as ninefold.

However, more research is needed to make curcumin fully

ready to be utilized effectively in a clinical setting.

In conclusion, our study was able to prove the efficacy

of intraperitoneal curcumin in vivo. Intraperitoneal curcu-

min was able to reduce uterine leiomyosarcoma cell growth

in vivo via the inhibition of mTOR activity that resulted in

the induction of apoptosis. Our study and analyses suggest

that, although promising, the bioavailability of curcumin

needs to be significantly improved for curcumin to be

effectively utilized clinically.

Acknowledgments We thank Ms. Emi Endo, Ms. Etsuko Oba, and

Ms. Etsuko Tomita for assistance in tissue processing and immuno-

histochemical analysis. This study was approved by the Ethics

Committee for animal research and was supported by a Grant-in-aid

from the Japanese Ministry of Education, Science, Sports, and Cul-

ture, Tokyo, Japan (23592430).

Conflict of interest The authors report no conflict of interest.

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