A polymeric nanoparticle formulation of curcumin in combination with sorafenib synergistically inhibits tumor growth and metastasis in an orthotopic model of human hepatocellular carcinoma

Accepted Manuscript A polymeric nanoparticle formulation of curcumin in combination with sorafenib synergistically inhibits tumor growth and metastasis in an orthotopic model of human hepatocellular carcinoma Bo Hu, Ding Sun, Chao Sun, Yun-Fan Sun, Hai-Xiang Sun, Qing-Feng Zhu, Xin-Rong Yang, Ya-Bo Gao, Wei-Guo Tang, Jia Fan, Anirban Maitra, Robert A. Anders, M.D., Ph.D., Yang Xu, M.D., Ph.D. PII:

S0006-291X(15)30731-2

DOI:

10.1016/j.bbrc.2015.10.031

Reference:

YBBRC 34715

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 23 September 2015 Accepted Date: 6 October 2015

Please cite this article as: B. Hu, D. Sun, C. Sun, Y.-F. Sun, H.-X. Sun, Q.-F. Zhu, X.-R. Yang, Y.B. Gao, W.-G. Tang, J. Fan, A. Maitra, R.A. Anders, Y. Xu, A polymeric nanoparticle formulation of curcumin in combination with sorafenib synergistically inhibits tumor growth and metastasis in an orthotopic model of human hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.10.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT TITLE PAGE

2

A polymeric nanoparticle formulation of curcumin in combination with

3

sorafenib synergistically inhibits tumor growth and metastasis in an orthotopic

4

model of human hepatocellular carcinoma

5

Bo Hu1*, Ding Sun1,6*, Chao Sun1*, Yun-Fan Sun1, Hai-Xiang Sun1, Qing-Feng Zhu2,4,

6

Xin-Rong Yang1, Ya-Bo Gao5, Wei-Guo Tang1, Jia Fan1,4*, Anirban Maitra3*，Robert

7

A. Anders2* and Yang Xu1*

8

1

9

University; Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of

SC

RI PT

1

M AN U

Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan

10

Education, Shanghai 200032, P. R. China

11

2

12

Liver Pathology, Baltimore, MD 21205, USA

13

3

14

Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

15

4

16

5

17

Shanghai 200032, P. R. China

18

6

19

University, Suzhou 215004, P. R. China.

20

* These authors contributed equally to this work.

TE D

The Johns Hopkins University School of Medicine, Division of Gastrointestinal and

EP

The Sol Goldman Pancreatic Cancer Research Center, Departments of Oncology,

AC C

Institute of Biomedical Sciences, Fudan University, Shanghai 200032, P. R. China Department of Radiation Oncology, Zhongshan Hospital, Fudan University,

Department of Hepatobiliary Surgery, First Affiliated Hospital of Soochow

21 1

ACCEPTED MANUSCRIPT 1 Corresponding authors:

3

Yang Xu, M.D., Ph.D., Liver Cancer Institute, Fudan University, 136 Yi Xue Yuan

4

Road, Shanghai 200032, P. R. China

5

Tel &Fax: +86-21-64037181; E-mail: [email protected]

6

RI PT

2

Robert A. Anders, M.D., Ph.D., The Johns Hopkins University School of Medicine,

8

Division of Gastrointestinal and Liver Pathology, Baltimore, MD 21205, USA

9

Tel: +1-410-955-3511; Fax: +1 410 614 0671; E-mail: [email protected]

M AN U

SC

7

10

Running title: NanoCurcumin and sorafenib inhibit orthotopic HCC

12

TE D

11

The precise word count:

14

Number of figures: 4

16 17 18

AC C

15

3895

EP

13

19 20 21 2

ACCEPTED MANUSCRIPT Summary

2

Curcumin, a yellow polyphenol extracted from the rhizome of turmeric root

3

(Curcuma longa) has potent anti-cancer properties in many types of tumors with

4

ability to reverse multidrug resistance of cancer cells. However, widespread clinical

5

application of this agent in cancer and other diseases has been limited due to its poor

6

aqueous solubility. The recent findings of polymeric nanoparticle formulation of

7

curcumin (NFC) have shown the potential for circumventing the problem of poor

8

solubility, however evidences for NFC’s anti-cancer and reverse multidrug resistance

9

properties are lacking. Here we provide models of human hepatocellular carcinoma

10

(HCC), the most common form of primary liver cancer, in vitro and in vivo to

11

evaluate the efficacy of NFC alone and in combination with sorafenib, a kinase

12

inhibitor approved for treatment of HCC. Results showed that NFC not only inhibited

13

the proliferation and invasion of HCC cell lines in vitro, but also drastically

14

suppressed primary tumor growth and lung metastases in vivo. Moreover, in

15

combination with sorafenib, NFC induced HCC cell apoptosis and cell cycle arrest.

16

Mechanistically, NFC and sorafenib synergistically down-regulated the expression of

17

MMP9 via NF-κB/p65 signaling pathway. Furthermore, the combination therapy

18

significantly decreased the population of CD133-positive HCC cells, which have been

19

reported as cancer initiating cells in HCC. Taken together, NanoCurcumin provides an

20

opportunity to expand the clinical repertoire of this agent. Additional studies utilizing

21

a combination of NanoCurcumin and sorafenib in HCC are needed for further clinical

22

development.

AC C

EP

TE D

M AN U

SC

RI PT

1

3

ACCEPTED MANUSCRIPT 1 2

Key words: Curcumin, Sorafenib, Hepatocellular carcinoma, NF-κB, MMP-9

AC C

EP

TE D

M AN U

SC

RI PT

3

4

ACCEPTED MANUSCRIPT Introduction

2

Liver cancer is the second most common cause of cancer death among men, and the

3

sixth leading cause of cancer death among women. Almost half of liver cancer cases

4

and deaths worldwide are estimated to have occurred in China and hepatocellular

5

carcinoma (HCC) accounts for 70% to 85% of liver cancers globally [1,2]. Because

6

tumors can develop resistance to chemotherapeutic agents, there is an urgent need for

7

the development of agents that can reverse drug-resistance and suppress proliferation

8

and metastasis of HCC without toxicity to normal cells [3].

M AN U

SC

RI PT

1

Sorafenib is a vascular endothelial growth factor receptor and multikinase

10

inhibitor, approved for the treatment of unresectable HCCs. This drug has been used

11

as a first-line treatment for advanced HCC. Although sorafenib can prolong median

12

survival time by almost 3 months in patients with late-stage HCC (10.7 vs. 7.9

13

months), its application is limited because of its high cost, partial effect on metastasis,

14

and severe adverse side-effects, including risk of hemorrhage [4,5].

EP

15

TE D

9

Curcumin

(1,7-bis(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione;

diferuloylmethane) is a diphenolic compound extracted from the rhizome of turmeric

17

(Curcuma longa), a plant grown in tropical Southeast Asia [6]. It has been used in the

18

treatment of anorexia, inflammation, and biliary and hepatic disorders. In traditional

19

Indian medicine, turmeric is also used to treat sinusitis and rheumatism [7], and recent

20

evidences suggests that curcumin have potential antitumor effects in colon, lung,

21

breast, pancreatic, and prostate cancers and can also reverse multidrug resistance in

AC C

16

5

ACCEPTED MANUSCRIPT cancer cells [8-15]. However, widespread clinical use of curcumin is limited by its

2

poor bioavailability in oral formulations [16]. In rat model, most of the curcumin

3

administered orally is excreted in feces, resulting in a low blood concentration of

4

curcumin. However, a nanoparticle-encapsulated formulation of curcumin (NFC) has

5

been shown to improve the solubility, bioavailability, and pharmacokinetic properties

6

of free curcumin [17]. It has also been shown to suppress the effects of the carcinogen,

7

diethylnitrosamine, and inhibit HCC growth [18]. In this study, we evaluated the

8

ability of NanoCurcumin [17] combined with sorafenib to suppress proliferation,

9

migration or metastasis of human HCC cells in vitro and in vivo, and investigated the

SC

M AN U

10

RI PT

1

potential mechanism underlying its effects.

EP AC C

12

TE D

11

6

ACCEPTED MANUSCRIPT Materials and Methods

2

Drug formulations

3

Polymeric encapsulated curcumin was prepared as described [17]. A stock solution of

4

sorafenib (Bayer Pharmaceutical Corporation) was prepared in 100% dimethyl

5

sulfoxide (DMSO). For in vitro experiments, working solutions were prepared by

6

diluting the stock solution with Dulbecco’s Modified Eagle Medium (DMEM) (final

7

DMSO concentration is 0.1%).

SC

RI PT

1

9

M AN U

8 Cell lines

The HCC cell line, Huh7, was supplied by the Japanese Cancer Research Resources

11

Bank. MHCCLM3 and MHCCLM3-RFP (red fluorescent protein) cells, human HCC

12

cell lines with high metastatic potential, were established in our lab [19,20]. The cells

13

were cultured in DMEM with high glucose supplemented with 10% fetal bovine

14

serum (Gibco BRL, Grand Island, NY).

EP

15

TE D

10

In vitro cell proliferation, wound-healing, and Matrigel invasion assays

17

Cell proliferation was assessed using the cell counting kit-8 (CCK8) assay (Dojindo

18

Laboratories, Kumamoto, Japan) according to the manufacture instruction [21]. The

19

cells were ttreated with 1% DMSO (control), 40 µM NFC, 10 µM sorafenib, or

20

combination treatment (40 µM NFC + 10 µM sorafenib). The wound healing assay

21

was performed as previously described [21]. The cells were washed with

AC C

16

7

ACCEPTED MANUSCRIPT phosphate-buffered saline (PBS) and treated with 1% DMSO, 40 µM NFC, 10 µM

2

sorafenib, or 40 µM NFC + 10 µM sorafenib for 48 hours. The in vitro invasion assay

3

was performed by using a Matrigel-coated filter (No. 3422, Corning, USA).

4

MHCCLM3 or Huh7 cells (2×104 cells/well) were seeded into the migration chamber

5

containing serum-free DMEM with 1 % DMSO, 40 µM NFC, 10 µM sorafenib, or 40

6

µM NFC + 10 µM sorafenib, and incubated for 24 hours at 37 °C. Cells that migrated

7

to the lower surface of the filters were stained and counted under a light microscope.

8

All assays were performed in triplicate.

M AN U

SC

RI PT

1

9

Cell cycle analysis and apoptosis assay

11

Huh7 and MHCCLM3 cells were seeded into 6-well plates, incubated overnight, and

12

then treated with 1% DMSO, 40 µM NFC, 10 µM sorafenib, or 40 µM NFC + 10 µM

13

sorafenib for 48 hours. For cell cycle analysis, the cells were fixed in 75 % ethanol for

14

1 hour at 4 °C, and stained with the DNA-binding dye propidium iodide (1 mg/mL)

15

and RNase (0.5 mg/mL) for 30 min at 37°C. Finally, the DNA changes in cells were

16

examined with a flow cytometer (Beckman cytomics FC500). For apoptosis assay, the

17

cells were stained using the Annexin-V-FITC/PI apoptosis detection kit (BD

18

Biosciences, USA) according to the manufacturer’s instructions. Each test was

19

performed in triplicate.

AC C

EP

TE D

10

20 21

Orthotopic xenograft models of HCC

8

ACCEPTED MANUSCRIPT MHCCLM3-RFP cells (1×106) in 0.2 mL serum-free culture medium were injected

2

subcutaneously into three 4- to 6-week old male athymic BALB/c nu/nu mice

3

(Shanghai Institute of Material Medical, Chinese Academy of Science). The mice

4

were housed at the Zhongshan Hospital of Fudan University and the experimental

5

protocol was approved by the Animal Welfare Committee of Fudan University.

6

RI PT

1

When the subcutaneous tumor reached 1 cm in diameter, it was minced into pieces (approximately 2 mm3) and then implanted into the livers of 24 mice.

8

mice were randomly divided into four groups according to treatment: 1) control (PBS,

9

100 µl, intraperitoneal injection), 2) NFC (1.56 g/kg, daily intraperitoneal injection), 3)

10

sorafenib (30 mg/kg, daily oral administration), and 4) NFC plus sorafenib

11

(administered as described for single agent treatments). After 4 weeks, the mice were

12

sacrificed by overdosage of pentobarbital, and the tumors were removed for

13

measurement

14

transcription-polymerase chain reaction (qRT-PCR), and immunohistochemical

15

staining. Primary tumor volumes were calculated using the formula V = 1/2a × b2,

16

where a is the longest tumor axis, and b is the shortest tumor axis. Each metastatic

17

tumor in the lungs was cryosectioned and observed under fluorescence microscopy

18

(for excitation of RFP at 584 nm). Finally, the lung sections were stained with

19

hematoxylin and eosin (H&E) to quantify metastatic foci.

TE D analysis

by

western

blot,

quantitative

reverse

AC C

EP

and

20 21

The

M AN U

SC

7

Western blot analysis

9

ACCEPTED MANUSCRIPT The concentration of protein extracted from the HCC cell lines and mouse tumor

2

tissue was determined by using the BCA Protein Assay Kit (Pierce, USA). Samples

3

were analyzed with primary antibodies against matrix metalloproteinase 9 (MMP-9;

4

AB911, R&D Systems, 1:1000), tissue inhibitor of metalloproteinases-1 (TIMP-1; No.

5

8946, Cell Signaling Technology, 1:1000), nuclear factor-kappa-light-chain-enhancer

6

of activated B cells (NF-κB; No. 3034, Cell Signaling Technology, 1:1000),

7

unphosphorylated extracellular signal-related kinase (ERK1/2; ab17942, Abcam,

8

1:1000), and phosphorylated ERK 1/2 (No. 9102, Cell Signaling Technology, 1:1000).

M AN U

SC

RI PT

1

9

Enzyme-linked immunosorbent assay (ELISA)

11

MHCCLM3 and Huh7 cells were treated with 1% DMSO, 40 µM NFC, 10 µM

12

sorafenib, or 40 µM NFC + 10 µM sorafenib for 48 hours.Samples were incubated

13

with a monoclonal anti-MMP-9 antibody (AB911, R&D Systems), and incubated with

14

an enzyme-linked polyclonal antibody specific for MMP-9 (KHC3061, Life

15

Technologies). After washing away the unbound conjugate, a substrate solution was

16

added to each well, and absorbance at 450 nm was determined using a microplate

17

reader (Bio-Rad). All measurements were performed in duplicate, and MMP-9

18

concentration was expressed in pg/mL.

AC C

EP

TE D

10

19 20

Immunohistochemistry

10

ACCEPTED MANUSCRIPT Liver tumor specimens from the in vivo studies were fixed in 10 % formalin. After

2

dehydration in graded ethanol, the specimens were embedded in paraffin, sectioned (4

3

µm thick), placed on coated glass slides, deparaffinized in xylene and ethanol, and

4

rehydrated through a graduated alcohol series to distilled water. The sections were

5

then placed in 0.01 M citrate buffer (pH 6.0) and heated 98-100 °C, cooling (5-10

6

min), repeated twice in a microwave for antigen retrieval. After incubation with

7

primary antibodies against MMP-9 (AB911, R&D Systems, 1:100) or CD133 (No.

8

130-090-422, Miltenyi Biotec, 1:300) for 60 minutes at room temperature or

9

overnight at 4°C, the sections were incubated with secondary antibodies. The tissues

10

were stained using 3, 3'-diaminobenzidine (DAB) peroxidase substrate, and images

11

were obtained using a Leica DMLA light microscope (Leica Microsystems, Wetzlar,

12

Germany).

13

TE D

M AN U

SC

RI PT

1

qRT-PCR

15

Total RNA was extracted from cell lines and frozen tumor specimens using Trizol

16

reagent (Invitrogen), and 1 µg total RNA was reverse transcribed using the

17

PrimeScript RT Reagent kit (Takara Bio, Tokyo, Japan). Real-time PCR was

18

performed using a SYBR PrimeScript RT-PCR Kit (Takara Bio) and ABI7300

19

instrument (Applied Biosystems) according to the manufacturer's instructions.

20

Three independent experiments were performed, and all reactions were performed in

21

triplicate with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal

AC C

EP

14

11

ACCEPTED MANUSCRIPT 1

control. Primer sequences for NF-κB, MMP-9, and GAPDH were as follows: NF-κB,

2

forward

3

CAAGGCTGGGGAATTAAACTGGGGCAACCCCC;

4

GCCAACTACGACACCGACGAC, reverse TTGGCCTTGGAAGATGAATGGA;

5

CD133

6

ACGCCTTGTCCTTGGTAGTGTTG;

7

GGCATCCTGGGCTACACTGA,

8

Relative mRNA levels were calculated according to the equation: 2-∆Ct [∆Ct = Ct

9

(target) – Ct (GAPDH)].

GGGGGTTGCCCCAGTTTAATTCCCCAGCCTTG,

10

RI PT

CTGGGGCTGCTGTTTATTATTCTG,

reverse

GAPDH

reverse

forward

SC

and

forward

GTGGTCGTTGAGGGCAATG.

M AN U

forward

MMP-9

reverse

Flow cytometry analysis of CD133 population

12

The anti-human CD133-PE antibody (No. 130-080-801, Miltenyi Biotec) was used

13

for immunofluorescent detection of CD133-expressing cells by flow cytometry, using

14

the isotype-matched mouse immunoglobulin (No. 130-092-212, Miltenyi Biotec) as a

15

control. Cells were incubated with the primary antibody in PBS containing 2% bovine

16

serum albumin and 0.1% sodium azide, and analyzed using a FACSCalibur™

17

cytometer (BD Biosciences).

EP

AC C

18

TE D

11

19

Statistical analysis

20

Statistical analyses were performed with SPSS 19.0 for Windows. Quantitative data

21

were presented as mean ± standard error (SE) of at least three independent

12

ACCEPTED MANUSCRIPT 1

experiments. Continuous data were analyzed by one-way ANOVA and Student’s t-test,

2

and categorical data were analyzed by Fisher’s exact test or chi-square test; P < 0.05

3

was considered significant.

RI PT

4

AC C

EP

TE D

M AN U

SC

5

13

ACCEPTED MANUSCRIPT Results

2

NanoCurcumin inhibits HCC cell proliferation and invasion in vitro

3

Results of the CCK8 assay showed that NanoCurcumin significantly inhibited the

4

viability of Huh7 and MHCCLM3 cells (P < 0.01; Fig. S1). In MHCCLM3 cells the

5

half maximal inhibitory concentration (IC50) of NanoCurcumin was 40 µM at 24

6

hours, 35 µM at 48 hours, and 33 µM at 72 hours, and in Huh7 cells, the IC50 of

7

NanoCurcumin was 74 µM (24 hours), 30 µM (48 hours), and 27 µM (72 hours).

8

Although combination treatment appeared to more strongly inhibit cell proliferation,

9

the inhibition was not statistically significant from that of NanoCurcumin or sorafenib

SC

M AN U

10

RI PT

1

alone (Fig. 1A).

To further determine the cellular effects of NanoCurcumin on HCC, we

12

performed wound healing and migration assays. We found that the wound healing

13

ability of combination treatment was at least 2-fold lower than that of sorafenib alone

14

(Fig. 1B-1C). Consistent with these results, NanoCurcumin substantially reduced the

15

migration of both MHCCLM3 and Huh7 cells in the Boyden chamber migration assay

16

compared with the control (P < 0.01), and the combination treatment reduced the

17

migration of MHCCLM3 cells (P < 0.05) (Fig. 1D).

EP

AC C

18

TE D

11

19

NanoCurcumin induces HCC cell apoptosis and cell cycle arrest

20

Results of flow cytometry showed that NanoCurcumin and/or sorafenib significantly

21

increased the percentages of early apoptotic, late apoptotic, and necrotic Huh7 and

14

ACCEPTED MANUSCRIPT MHCCLM3 cells, and induction of apoptosis by combination treatment was

2

significantly higher that of NanoCurcumin or sorafenib alone (all P < 0.05; Fig. 2A,

3

2B, S2A and S2B). In experiments using MHCCLM3 cells, more necrotic cells were

4

detected after combination treatment than after treatment with NanoCurcumin or

5

sorafenib alone (P < 0.01) (Fig. Fig. 2A, 2B, S2A and S2B ).

RI PT

1

In addition, NanoCurcumin and/or sorafenib treatments induced G2/M arrest in

7

both cell lines (all P < 0.01), but the proportion of cells in G2/M arrest was higher

8

after combination treatment compared with single agent treatments (Fig. 2C , 2D,

9

S2C and S2D).

M AN U

SC

6

10

NanoCurcumin inhibits in vivo progression and metastasis of HCC cells

12

One week after subcutaneous and orthotopic tumor implantation, athymic mice were

13

treated with PBS (control), NanoCurcumin, sorafenib, or both NanoCurcumin and

14

sorafenib. After 4 weeks of treatment, the subcutaneous and orthotopic liver

15

xenografts were harvested (Fig. 2E). No adverse effects (e.g., morbidity, mortality,

16

body weight changes) were observed in any of these groups. Volumes of the

17

subcutaneous tumors were smaller in the treated mice (P < 0.05; Fig. 2F). Although

18

combination treatment appeared to exert an effect in reducing orthotopic tumor

19

weight compared with NanoCurcumin alone (0.41 ± 0.14 vs. 0.75 ± 0.96 g) or

20

sorafenib alone (0.41 ± 0.14 vs. 0.48 ± 0.11 g), these differences were not statistically

21

significant (P > 0.05; Table 1). We then examined the mice for metastatic tumors in

AC C

EP

TE D

11

15

ACCEPTED MANUSCRIPT the diaphragm, peritoneal cavity, lymph nodes, and visceral organs, and found that

2

tumors formed primarily in the lungs (Fig. 2G). Tumors were detected in all mice in

3

the control group but in only 50% of the NanoCurcumin -treated mice (P > 0.05;

4

Table 1). However, combination treatment showed the best therapeutic efficacy,

5

significantly reducing lung metastatic tumors compared with control treatment (16.7%

6

vs. 100%; P = 0.015).

RI PT

1

SC

7

NanoCurcumin in combination with sorafenib regulates MMP-9 and its

9

endogenous Inhibitors in vivo and in vitro.

M AN U

8

To investigate the mechanism by which NanoCurcumin and sorafenib inhibit HCC

11

metastasis, we evaluated expression of MMP-9, an important marker of HCC

12

migration [22,23]. Our results demonstrated that combination treatment decreased

13

MMP-9 mRNA levels both in vitro and in vivo (Fig. 3A and 4A).

14

western blot and ELISA results demonstrated that combination treatment decreased

15

MMP-9 and increased TIMP-1, the inhibitor of MMP-9, compared with single agent

16

treatments

17

immunohistochemical staining (Fig. 3A).

Furthermore, both

EP

AC C

18

TE D

10

(Fig.

3D,

3E,

4D,

4E).

Similar

results

were

obtained

by

19

NanoCurcumin in combination with sorafenib inhibits MMP-9 expression by

20

inactivating NF-κB /p65 in vivo and in vitro.

16

ACCEPTED MANUSCRIPT In human head and neck squamous cell carcinoma cells, curcumin has been shown to

2

suppress MMP-9 expression through modulation of NF-κB activity [24]. We therefore

3

investigated the effects of NanoCurcumin combined with sorafenib on NF-κB activity

4

in MHCCLM3 and Huh7 cells. Our results demonstrated that p65 was significantly

5

elevated in the cytoplasm but was dramatically decreased in the nucleus of cells when

6

treated with both NanoCurcumin and sorafenib compared with single agent treatments

7

(Fig. 3E). In addition, combination treatment significantly suppressed p65 mRNA

8

levels (Fig. 3B). Similar results were obtained in vivo (Fig. 4B, 4E). In addition,

9

western blot analysis showed that NanoCurcumin and sorafenib combination

10

treatment decreased phosphorylation of ERK1/2 both in vitro and in vivo (Fig. 3E

11

and 4E).

SC

M AN U

TE D

12

RI PT

1

NanoCurcumin in combination with sorafenib decreases CD133-positive HCC

14

cells in vivo and in vitro.

15

NanoCurcumin in combination with sorafenib downregulated CD133 mRNA levels

16

both in vivo and in vitro (all P < 0.01; Fig. 3C and 4C), and significantly decreased

17

the population of CD133-positive cells compared with either NanoCurcumin or

18

sorafenib alone (P < 0.05; Fig. 4C and S4). Results of flow cytometry showed that

19

the percentage of CD133-positive MHCCLM3 cells was decreased by NanoCurcumin

20

alone (13.80 ± 0.63% vs. 20.81 ± 1.07%; P < 0.01) and sorafenib alone (9.42 ± 0.52%

21

vs. 20.81 ± 1.07%; P < 0.01) compared with control (Fig. 4C and S4). However,

AC C

EP

13

17

ACCEPTED MANUSCRIPT combination treatment was most effective in decreasing CD133 marker expression in

2

both MHCCLM3 (4.82 ± 1.22%) and Huh7 (5.46 ± 0.68%) cells (Fig. 4C and S4).

3

Protein levels of CD133, as assessed by immunohistochemistry, were consistent with

4

our flow cytometry results (Fig. S4C).

RI PT

1

AC C

EP

TE D

M AN U

SC

5

18

ACCEPTED MANUSCRIPT Discussion

2

The 5-year survival rate of patients with HCC remains lower than 15 % because of

3

metastasis and recurrence [25,26]. Thus, there is an urgent need for drugs with

4

anti-metastatic effects. Natural products have proven to be important sources of

5

cancer chemotherapeutic and chemopreventive agents, with 70% of current anticancer

6

drugs being derived from natural products [27,28].

RI PT

1

A nanoparticle-encapsulated formulation of curcumin overcomes the poor water

8

solubility of free curcumin and improves its therapeutic index. We found that

9

NanoCurcumin treatment effectively decreased cell proliferation, migration and

10

invasion of HCC cells. These results were consistent with the effects of high doses of

11

free curcumin in other HCC cell lines (HEP3B, SK-Hep-1, SNU449) [29]. Previous

12

studies have reported that NanoCurcumin synergistically enhances the antitumor

13

effects of doxorubicin [30-32]. Therefore, in this study we investigated the potential

14

synergistic effects of NanoCurcumin with the a kinase inhibitor, sorafenib, in the

15

treatment of HCC. Our results show that the combination of NanoCurcumin and

16

sorafenib produces stronger antitumor effects on HCC than either NanoCurcumin or

17

sorafenib alone. Combination treatment inhibited HCC cell

18

and increased cell apoptosis in vitro and in vivo, dramatically decreasing the number

19

of pulmonary metastases. These results indicate the ability of NanoCurcumin to

20

attenuate HCC progression and metastasis.

AC C

EP

TE D

M AN U

SC

7

19

migration and invasion,

ACCEPTED MANUSCRIPT To better understand the mechanism underlying the anticancer effects of

2

NanoCurcumin, we evaluated its effects on MMP-9, which facilitates tumor

3

metastasis [33,34]. Previous reports have indicated free curcumin suppresses MMP-9

4

expression in HCC through an unknown mechanism [35,36]. Because NF-κB is a key

5

transcription factor involved in MMP-9 expression, we investigated the effect of

6

NanoCurcumin on NF-κB signaling and found that NanoCurcumin inhibited the

7

nuclear translocation of the active form of NF-κB, phosphorylated p65, thereby

8

decreasing MMP-9 expression and increasing TIMP-1 expression [37,38]. In addition,

9

NanoCurcumin inhibited the phosphorylation of ERK1/2, a key downstream effector

10

of the mitogen-activated protein kinase (MAPK)/ERK pathway. The MAPK pathway

11

is often constitutively active in HCC, leading to overexpression of genes that promote

12

cell proliferation.

TE D

M AN U

SC

RI PT

1

Liang et al. reported that use of the curcumin analog EF24 could overcome

14

sorafenib resistance through von Hippel Lindau tumor suppressor-dependent

15

degradation of hypoxia-inducible factor 1-α and inactivation of NF-κB [39]. This may

16

partly explain why combining sorafenib with NanoCurcumin enhances its anticancer

17

effects.

18

Raf/mitogen-activated protein extracellular kinase/ERK signaling at the level of Raf

19

kinase [40]. Its combination with NanoCurcumin may act synergistically to

20

downregulate

21

phosphorylation and NF-κB DNA-binding activities.

AC C

EP

13

Sorafenib

MMP-9

suppresses

through

ERK

the

phosphorylation

simultaneous

20

inhibition

by

of

targeting

ERK1/2

ACCEPTED MANUSCRIPT Recent studies have suggested that CD133 is a biomarker for cancer stem cells in

2

HCC, and the malignant characteristics of CD133-positive human HCC cells are

3

regulated by MMPs [41-43]. In view of these findings, we evaluated the effects of

4

NanoCurcumin on the CD133-positive population of HCC cells. The results showed

5

that NanoCurcumin significantly decreased CD133-positive cells, and combining

6

NanoCurcumin with sorafenib produced the strongest effects. However, it remains

7

unclear how NanoCurcumin decreases the expression of CD133 or how combination

8

treatment enhances this activity.

M AN U

SC

RI PT

1

In summary, our results indicate that combination therapy with NanoCurcumin

10

and sorafenib represents a promising strategy for the treatment of HCC and needs

11

further clinical investigation.

AC C

EP

TE D

9

21

ACCEPTED MANUSCRIPT 1 Acknowledgments:

3

Supported by National Key Sci-Tech Project (2013ZX10002011-004), National

4

Natural Science Foundation of China (81372317, 81071661 and 81302100), the

5

Shanghai Pujiang Scholar award (13PJD007), National Institute of Health USA (DK

6

080736) and Specialized Research Fund for the Doctoral Program of Higher

7

Education of China (20120071120068).

AC C

EP

TE D

M AN U

SC

RI PT

2

22

ACCEPTED MANUSCRIPT 1

Disclosure Statement:

2

The authors disclose no conflicts of interest.

AC C

EP

TE D

M AN U

SC

RI PT

3

23

ACCEPTED MANUSCRIPT References

2

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, et al. (2011) Global cancer

3

statistics. CA Cancer J Clin 61: 69-90.

4

2. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, et al. (2010) Estimates of

5

worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 127:

6

2893-2917.

7

3. Yallapu MM, Jaggi M, Chauhan SC (2012) Curcumin nanoformulations: a future

8

nanomedicine for cancer. Drug Discov Today 17: 71-80.

9

4. Darvesh AS, Aggarwal BB, Bishayee A (2012) Curcumin and liver cancer: a review.

SC

RI PT

1

Curr Pharm Biotechnol 13: 218-228.

11

5. Yau T, Chan P, Ng KK, Chok SH, Cheung TT, et al. (2009) Phase 2 open-label

12

study of single-agent sorafenib in treating advanced hepatocellular carcinoma in a

13

hepatitis B-endemic Asian population: presence of lung metastasis predicts poor

14

response. Cancer 115: 428-436.

15

6. Maheshwari RK, Singh AK, Gaddipati J, Srimal RC (2006) Multiple biological

16

activities of curcumin: a short review. Life Sci 78: 2081-2087.

17

7. Singh S (2007) From exotic spice to modern drug? Cell 130: 765-768.

18

8. Sa G, Das T (2008) Anti cancer effects of curcumin: cycle of life and death. Cell

19

Div 3: 14.

20

9. Kwon Y, Magnuson BA (2009) Age-related differential responses to

21

curcumin-induced apoptosis during the initiation of colon cancer in rats. Food Chem

22

Toxicol 47: 377-385.

23

10. Yang CL, Liu YY, Ma YG, Xue YX, Liu DG, et al. (2012) Curcumin blocks small

24

cell lung cancer cells migration, invasion, angiogenesis, cell cycle and neoplasia

25

through Janus kinase-STAT3 signalling pathway. PLoS One 7: e37960.

26

11. Sinha D, Biswas J, Sung B, Aggarwal BB, Bishayee A (2012) Chemopreventive

27

and chemotherapeutic potential of curcumin in breast cancer. Curr Drug Targets 13:

28

1799-1819.

29

12. Wang Z, Zhang Y, Banerjee S, Li Y, Sarkar FH (2006) Notch-1 down-regulation

AC C

EP

TE D

M AN U

10

24

ACCEPTED MANUSCRIPT by curcumin is associated with the inhibition of cell growth and the induction of

2

apoptosis in pancreatic cancer cells. Cancer 106: 2503-2513.

3

13. Lev-Ari S, Zinger H, Kazanov D, Yona D, Ben-Yosef R, et al. (2005) Curcumin

4

synergistically potentiates the growth inhibitory and pro-apoptotic effects of celecoxib

5

in pancreatic adenocarcinoma cells. Biomed Pharmacother 59 Suppl 2: S276-280.

6

14. Khor TO, Keum YS, Lin W, Kim JH, Hu R, et al. (2006) Combined inhibitory

7

effects of curcumin and phenethyl isothiocyanate on the growth of human PC-3

8

prostate xenografts in immunodeficient mice. Cancer Res 66: 613-621.

9

15. Deeb D, Jiang H, Gao X, Hafner MS, Wong H, et al. (2004) Curcumin sensitizes

SC

RI PT

1

10

prostate

11

ligand/Apo2L by inhibiting nuclear factor-kappaB through suppression of

12

IkappaBalpha phosphorylation. Mol Cancer Ther 3: 803-812.

13

16. Sharma RA, McLelland HR, Hill KA, Ireson CR, Euden SA, et al. (2001)

14

Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients

15

with colorectal cancer. Clin Cancer Res 7: 1894-1900.

16

17. Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, et al. (2007) Polymeric

17

nanoparticle-encapsulated curcumin ("nanocurcumin"): a novel strategy for human

18

cancer therapy. J Nanobiotechnology 5: 3.

19

18. Ghosh D, Choudhury ST, Ghosh S, Mandal AK, Sarkar S, et al.

20

(2012)Nanocapsulated

21

diethylnitrosamine induced hepatocellular carcinoma in rat. Chem Biol Interact 195:

22

206-214.

23

19. Li Y, Tian B, Yang J, Zhao L, Wu X, et al. (2004) Stepwise metastatic human

24

hepatocellular carcinoma cell model system with multiple metastatic potentials

25

established through consecutive in vivo selection and studies on metastatic

26

characteristics. J Cancer Res Clin Oncol 130: 460-468.

27

20. Ye QH, Qin LX, Forgues M, He P, Kim JW, et al. (2003) Predicting hepatitis B

28

virus-positive metastatic hepatocellular carcinomas using gene expression profiling

cells

to

tumor

necrosis

factor-related

apoptosis-inducing

EP

TE D

M AN U

cancer

oral

chemopreventive

formulation

against

AC C

curcumin:

25

ACCEPTED MANUSCRIPT and supervised machine learning. Nat Med 9: 416-423.

2

21. Yang XR, Xu Y, Yu B, Zhou J, Li JC, et al. (2009) CD24 is a novel predictor for

3

poor prognosis of hepatocellular carcinoma after surgery. Clin Cancer Res 15:

4

5518-5527.

5

22. El Tayebi HM, Salah W, El Sayed IH, Salam EM, Zekri AR, et al. (2011)

6

Expression of insulin-like growth factor-II, matrix metalloproteinases, and their tissue

7

inhibitors as predictive markers in the peripheral blood of HCC patients. Biomarkers

8

16: 346-354.

9

23. Qin LX, Tang ZY (2004) Recent progress in predictive biomarkers for metastatic

10

recurrence of human hepatocellular carcinoma: a review of the literature. J Cancer

11

Res Clin Oncol 130: 497-513.

12

24. Aggarwal S, Takada Y, Singh S, Myers JN, Aggarwal BB (2004) Inhibition of

13

growth and survival of human head and neck squamous cell carcinoma cells by

14

curcumin via modulation of nuclear factor-kappaB signaling. Int J Cancer 111:

15

679-692.

16

25. Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J

17

Clin 62: 10-29.

18

26. Rahbari NN, Mehrabi A, Mollberg NM, Muller SA, Koch M, et al. (2011)

19

Hepatocellular carcinoma: current management and perspectives for the future. Ann

20

Surg 253: 453-469.

21

27. Pezzuto JM (1997) Plant-derived anticancer agents. Biochem Pharmacol 53:

22

121-133.

23

28. Newman DJ, Cragg GM, Holbeck S, Sausville EA (2002) Natural products and

24

derivatives as leads to cell cycle pathway targets in cancer chemotherapy. Curr Cancer

25

Drug Targets 2: 279-308.

26

29. Ning L, Wentworth L, Chen H, Weber SM (2009) Down-regulation of Notch1

27

signaling inhibits tumor growth in human hepatocellular carcinoma. Am J Transl Res

28

1: 358-366.

AC C

EP

TE D

M AN U

SC

RI PT

1

26

ACCEPTED MANUSCRIPT 30. Cheng CY, Lin YH, Su CC (2010) Curcumin inhibits the proliferation of human

2

hepatocellular carcinoma J5 cells by inducing endoplasmic reticulum stress and

3

mitochondrial dysfunction. Int J Mol Med 26: 673-678.

4

31. Jiang J, Jin H, Liu L, Pi J, Yang F, et al. (2013) Curcumin disturbed cell-cycle

5

distribution of HepG2 cells via cytoskeletal arrangement. Scanning 35: 253-260.

6

32. Meiyanto E, Putri DD, Susidarti RA, Murwanti R, Sardjiman, et al. (2014)

7

Curcumin and its analogues (PGV-0 and PGV-1) enhance sensitivity of resistant

8

MCF-7 cells to doxorubicin through inhibition of HER2 and NF-kB activation. Asian

9

Pac J Cancer Prev 15: 179-184.

SC

RI PT

1

33. Littlepage LE, Sternlicht MD, Rougier N, Phillips J, Gallo E, et al. (2010) Matrix

11

metalloproteinases contribute distinct roles in neuroendocrine prostate carcinogenesis,

12

metastasis, and angiogenesis progression. Cancer Res 70: 2224-2234.

13

34. Yang P, Yuan W, He J, Wang J, Yu L, et al. (2009) Overexpression of EphA2,

14

MMP-9, and MVD-CD34 in hepatocellular carcinoma: Implications for tumor

15

progression and prognosis. Hepatol Res 39: 1169-1177.

16

35. Lin LI, Ke YF, Ko YC, Lin JK (1998) Curcumin inhibits SK-Hep-1 hepatocellular

17

carcinoma cell invasion in vitro and suppresses matrix metalloproteinase-9 secretion.

18

Oncology 55: 349-353.

19

36. Ohashi Y, Tsuchiya Y, Koizumi K, Sakurai H, Saiki I (2003) Prevention of

20

intrahepatic metastasis by curcumin in an orthotopic implantation model. Oncology

21

65: 250-258.

22

37. Takeshita H, Yoshizaki T, Miller WE, Sato H, Furukawa M, et al. (1999) Matrix

23

metalloproteinase 9 expression is induced by Epstein-Barr virus latent membrane

24

protein 1 C-terminal activation regions 1 and 2. J Virol 73: 5548-5555.

25

38. Rangaswami H, Bulbule A, Kundu GC (2004) Nuclear factor-inducing kinase

26

plays a crucial role in osteopontin-induced MAPK/IkappaBalpha kinase-dependent

27

nuclear factor kappaB-mediated promatrix metalloproteinase-9 activation. J Biol

28

Chem 279: 38921-38935.

AC C

EP

TE D

M AN U

10

27

ACCEPTED MANUSCRIPT 1

39. Liang Y, Zheng T, Song R, Wang J, Yin D, et al. (2013) Hypoxia-mediated

2

sorafenib resistance can be overcome by EF24 through Von Hippel-Lindau

3

suppressor-dependent HIF-1alpha inhibition in hepatocellular carcinoma. Hepatology

4

57: 1847-1857.

5

40. Liu L, Cao Y, Chen C, Zhang X, McNabola A, et al. (2006) Sorafenib blocks the

6

RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell

7

apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 66:

8

11851-11858.

9

41. Kohga K, Tatsumi T, Takehara T, Tsunematsu H, Shimizu S, et al. (2010)

10

Expression of CD133 confers malignant potential by regulating metalloproteinases in

11

human hepatocellular carcinoma. J Hepatol 52: 872-879.

12

42. Hou Y, Zou Q, Ge R, Shen F, Wang Y (2012) The critical role of

13

CD133(+)CD44(+/high) tumor cells in hematogenous metastasis of liver cancers. Cell

14

Res 22: 259-272.

15

43. Ma S, Chan KW, Hu L, Lee TK, Wo JY, et al. (2007) Identification and

16

characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology

17

132: 2542-2556.

21

RI PT

SC

M AN U

TE D

AC C

20

EP

18 19

tumor

22

Figure Legends

23

Figure 1. Effect of NanoCurcumin and/or sorafenib (SO) on cell proliferation,

24

migration and invasion in vitro.

25

(A) Viability of Huh7 and MHCCLM3 cells was decreased by 48-hour treatment with

26

NanoCurcumin (40 µM) and/or SO (10 µM). (B,C) The wound healing abilities of

28

ACCEPTED MANUSCRIPT Huh7 and MHCCLM3 cells were evaluated after NanoCurcumin and/or SO treatment.

2

(D) To determine effects on invasion and migration, Huh7 and MHCCLM3 cells were

3

seeded onto Matrigel-coated Boyden chamber insert containing NanoCurcumin (40

4

µmol/L), SO (10 µmol/L), or NanoCurcumin (40 µmol/L) + SO (10 µmol/L). The

5

cells were stained and quantified after 8 hours. Results are expressed as mean ± SE of

6

at least three independent experiments. *P < 0.05, #P < 0.01 compared with vehicle

7

control. Magnification, ×100 (D, E). Scale bar, 200 µm.

SC

RI PT

1

9

M AN U

8

Figure 2. Effect of NanoCurcumin on tumor growth and metastasis of MHCCLM3-RFP cells in vitro and in vivo.

11

(A) Huh7 cells and (B) MHCCLM3 cells were treated with NanoCurcumin (40

12

µmol/L), SO (10 µmol/L), or NanoCurcumin (40 µmol/L) + SO (10 µmol/L) for 48

13

hours and analyzed by flow cytometry. The X-axis represents Annexin V, and the

14

Y-axis represents propidium iodide. Cell cycle analysis of (C) Huh7 cells and (D)

15

MHCCLM3 was assessed by flow cytometry. The Y-axis represents number of cells,

16

and the X-axis represents DNA content as fluorescence intensity of propidium iodide

17

staining. Results are expressed as mean ± SE of three independent experiments, *P <

18

0.05, #P < 0.01 compared with controls. (E) One week after MHCCLM3-RFP cell

19

implantation, male athymic BALB/c nu/nu mice underwent 28-day treatment with

20

NanoCurcumin (1.56 g/kg daily), sorafenib (SO, 30 mg/kg daily), or both.

21

Representative orthotopic and subcutaneous tumor xenografts for each treatment

AC C

EP

TE D

10

29

ACCEPTED MANUSCRIPT group. (F) Tumor volumes of treated mice were smaller than those of untreated mice

2

(*P < 0.05, #P < 0.01). (G) Left panel: hematoxylin and eosin staining of a metastatic

3

nodule in the lung; magnification of the selected areas showing number of tumor cells

4

within a single nodule. Right panel: representative image of metastasis in the lung

5

visualized as fluorescence of MHCCLM3-RFP cells. Flow cytometry analysis of (H)

6

Huh7 cells and (I) MHCCLM3 cells showed that NanoCurcumin and/or sorafenib

7

significantly decreased CD133-positive cells in vitro. Results expressed as mean ± SE

8

of at least three independent experiments.

M AN U

SC

RI PT

1

9

Figure 3. Effect of NanoCurcumin on NF-κB/p65, MMP-9, and CD133 in vitro.

11

Huh7 and MHCC97L cell lines were treated with NanoCurcumin (40 µM), sorafenib

12

(SO, 10 µM), or NanoCurcumin (40 µM) + SO (10 µM) for 48 hours. Results of

13

qRT-PCR showing mRNA levels of (A) MMP-9, (B) p65, and (C) CD133. Protein

14

levels of MMP-9, p65 (whole cell, cytoplasm and nucleus), TIMP-1, ERK1/2 (total

15

and phosphorylated), as determined by (D) ELISA and (E) western blot analysis.

EP

AC C

16

TE D

10

17

Figure 4. Mechanism of action of NanoCurcumin in HCC xenograft model.

18

After 28-day treatment with NanoCurcumin and/or sorafenib (SO), mRNA levels of

19

(A) MMP-9, (B) p65, and (C) CD133 levels in orthotopic tumors were determined by

20

qRT-PCR. Protein levels of MMP-9, p65 (whole cell, cytoplasm, and nucleus),

21

TIMP-1, ERK1/2 (total and phosphorylated), as determined by (D) ELISA and (E)

30

ACCEPTED MANUSCRIPT 1

western blot. Results are expressed as mean ± SE of at least three independent

2

experiments. *P < 0.05, #P < 0.01 compared with controls. Magnification, ×400 (D).

3

Scale bar, 200 µm.

RI PT

4

Figure S1. Effect of NanoCurcumin and/or sorafenib (SO) on cell proliferation in

6

vitro.

7

(A) Results of the CCK8 assay showed that NanoCurcumin inhibited proliferation of

8

Huh7 and MHCCLM3 cells.

9

Figure S2. Effect of NanoCurcumin and/or sorafenib (SO) on cell apoptosis and

M AN U

SC

5

cell cycle arrest in vitro.

11

(A) Huh7 cells and (B) MHCCLM3 cells were treated with NanoCurcumin (40

12

µmol/L), SO (10 µmol/L), or NanoCurcumin (40 µmol/L) + SO (10 µmol/L) for 48

13

hours and analyzed by flow cytometry. The X-axis represents Annexin V, and the

14

Y-axis represents propidium iodide. Cell cycle analysis of (C) Huh7 cells and (D)

15

MHCCLM3 was assessed by flow cytometry. The Y-axis represents number of cells,

16

and the X-axis represents DNA content as fluorescence intensity of propidium iodide

17

staining.

18

Figure S3. Effect of NanoCurcumin on MMP-9 in vivo.

19

(A) Immunohistochemical staining of orthotopic tumors for MMP-9.

20

Figure S4. Effect of NanoCurcumin on CD133 in vitro and in vivo.

AC C

EP

TE D

10

31

ACCEPTED MANUSCRIPT Flow cytometry analysis of (A) Huh7 cells and (B) MHCCLM3 cells showed that

2

NanoCurcumin and/or sorafenib significantly decreased CD133-positive cells in vitro.

3

Results expressed as mean ± SE of at least three independent experiments. (C)

4

Immunohistochemical staining of orthotopic tumors for CD133. *P < 0.05, # P <0.01

5

compared with controls.

RI PT

1

AC C

EP

TE D

M AN U

SC

6

32

ACCEPTED MANUSCRIPT Summary of orthotopic xenograft liver tumor model. SO

NFC+SO

6/6 100% 1.53±0.48

3/6 50% 0.75±0.96

4/6 66.7% 0.48±0.11

1/6 16.7% 0.41±0.14

M AN U TE D EP AC C

P value(NFC vs Control)

P value(SO vs Control)

P value(NF C+SO vs Control)

0.182 0.001

0.455 <0.001

0.015 <0.001

RI PT

NFC

SC

Pulmonary metastasis Percentage Tumor weight (g)

Control

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights 1． Polymeric nanoparticle formulation of curcumin not only inhibited the proliferation and invasion of HCC cell lines in vitro, but also drastically

RI PT

suppressed primary tumor growth and lung metastases in vivo. 2． In combination with sorafenib, NanoCurcumin induced HCC cell apoptosis and cell cycle arrest.

M AN U

MMP9 via NF-κB/p65 signaling pathway.

SC

3． NanoCurcumin and sorafenib synergistically down-regulated the expression of

4． NanoCurcumin and sorafenib significantly decreased the population of CD133-positive HCC cells.

5． NanoCurcumin provides an opportunity to expand the clinical repertoire of

TE D

curcumin. Additional studies utilizing a combination of NanoCurcumin and

AC C

EP

sorafenib in HCC are needed for further clinical development.