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Liquiritigenin Potentiates the Inhibitory Effects ofCisplatin on Invasion and Metastasis Via DownregulationMMP-2/9 and PI3 K/AKT Signaling Pathway in B16F10Melanoma Cells and Mice ModelHui Shia, Yuchen Wua, Yang Wanga, Ming Zhoua, Shu Yana, Zhimin Chena, Dawei Gua &Yunqing Caiaa Department of Nutrition and Food Hygiene, School of Public Health, Nanjing MedicalUniversity, Nanjing, ChinaPublished online: 15 May 2015.

To cite this article: Hui Shi, Yuchen Wu, Yang Wang, Ming Zhou, Shu Yan, Zhimin Chen, Dawei Gu & Yunqing Cai (2015)Liquiritigenin Potentiates the Inhibitory Effects of Cisplatin on Invasion and Metastasis Via Downregulation MMP-2/9and PI3 K/AKT Signaling Pathway in B16F10 Melanoma Cells and Mice Model, Nutrition and Cancer, 67:5, 761-770, DOI:10.1080/01635581.2015.1037962

To link to this article: http://dx.doi.org/10.1080/01635581.2015.1037962

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Liquiritigenin Potentiates the Inhibitory Effects of Cisplatinon Invasion and Metastasis Via Downregulation MMP-2/9and PI3 K/AKT Signaling Pathway in B16F10 Melanoma Cellsand Mice Model

Hui Shi, Yuchen Wu, Yang Wang, Ming Zhou, Shu Yan, Zhimin Chen,Dawei Gu, and Yunqing CaiDepartment of Nutrition and Food Hygiene, School of Public Health,

Nanjing Medical University, Nanjing, China

Liquiritigenin (LQ) is a flavanone extracted from glycyrrhizae.Previous studies have demonstrated that LQ possessesantimigration properties in HELA and A549 cells. The presentresearch, as an extension of our earlier ones, investigated whetherLQ can enhance the antimigration and antiinvasion effect of cis-diamine dichloroplatinum (CDDP) in B16F10 melanoma cell. Thedata indicated that LQ (25, 50, 100, 200mM) combined with CDDP(2 mM) significantly reduced B16F10 cell viability compared toCDDP (2 mM)-treated only. The different doses of LQ combinedwith CDDP significantly suppressed cell migration (21.5%, 49.6%,75.6%) and cell invasion (26.2%, 51.4%, 69.5%) compared withCDDP-treated alone, suggesting that LQ enhance the inhibitionaction of CDDP on cell migration and invasion. Moreover, LQ/CDDP combination led to the downregulation of proteinexpression of MMP-2/9, PI3 K, p-AKT, and upregulated PTENprotein level that play an important role in tumor metastasisprogression. Further study demonstrated the enhancement effectof LQ on CDDP suppressing lung metastasis in a mice model beinginoculated by the B16F10 melanoma cells. In conclusion, theresults suggested that LQ plays an intensive role on CDDPsuppressing invasion and metastasis through regulating thePI3 K/AKT signal pathway and suppressing the protein expressionofMMP-2/9.

INTRODUCTION

Cancer, one of the major public health problems in the

world, is severely endangering human health. It is a leading

cause of death worldwide, accounting for 7.6 million deaths

(around 13% of all deaths) in 2008 (1). Metastases, one of

the biological characteristics of malignant tumor, are the

major cause of death from cancer (2). It is all known that can-

cer can be cured if diagnosed and treated early, but with the

development of the diseases, once metastasis has occurred,

they are rarely effectual with treatment. This is particularly

true for melanoma that is a common cancer with highly

aggressive and metastatic properties (3). According to a

World Health Organization report, there are an estimated

132000 melanoma skin cancers and an estimated 48000

deaths from malignant melanoma globally each year (4). And

50–70% of Stage III patients with melanoma die of metastasis

within 5 yr after the initial diagnosis (5).

Tumor metastasis is multifactorial and multistep process

which involves the local invasion intravasation, transport

interaction with blood components, cell adhesion, extravasa-

tion migration, and growth at a new organ (6). In this process,

degrading the extracellular matrix (ECM), which is the natural

barrier of tumor invasion and metastasis, is the first important

step. According to many investigates, the matrix metallopro-

teinase (MMPs), particularly MMP-2 and MMP-9, are the

principal enzymes in the ECM degradation (7). Moreover, the

MMPs are overexpressed in various malignant tumors and

their over-expression is related to tumor invasion and metasta-

sis (8). Therefore, MMPs and their action mechanisms have

been the focus of intense research interest.

A major mechanism considered pivotal in cancer progres-

sion is the PI3 K/AKT intracellular signal transduction path-

way. It plays an important role in cells metastasis and is

constitutively activated by affecting the downstream impact

factor (9). Some reports suggest that the abnormal activation

of AKT promote cancer cell invasion and metastasis (10,11).

In melanoma, the low expression of PTEN cannot be

restrained effectively the abnormal activation of PI3 K/AKT

Submitted 05 November 2013; accepted in final form 23 March2015.

Address correspondence to Yunqing Cai, Department of Nutritionand Food Hygiene, School of Public Health, Nanjing Medical Univer-sity, 818 East Tianyuan Road, Nanjing 211166, China. E-mail:cai2941@163.comColor versions of one or more figures in this article can be found

online at www.tandfonline.com/hnuc.

761

Nutrition and Cancer, 67(5), 761–770

Copyright � 2015, Taylor & Francis Group, LLC

ISSN: 0163-5581 print / 1532-7914 online

DOI: 10.1080/01635581.2015.1037962

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pathway, which leads to tumor metastasis (12). Furthermore,

multiple in vitro studies demonstrate that the activity of MMP-

2 and MMP-9 are coordinately regulated by PI3 K/AKT signal

pathway (11,13). Therefore, effective inhibition of abnormal

activation of the PI3 K/AKT signal pathway is a method for

the treatment of tumors. In the clinical, cisplatin [cis-diamine

dichloroplatinum (CDDP)] has been one of the main chemo-

therapy drugs for defense against tumors including ovarian,

lung cancers and melanoma (14,15). Unfortunately, the treat-

ment effect is subject to its dose-limiting toxic side effects.

Thus, it is necessary to establish effective strategy for its treat-

ment with few harmful side effects.

In recent years, an increasing number of research have

focused on phytochemicals, which had been applied to the

field of cancer research. A prominent example of the phyto-

chemicals is flavonoids compounds. Flavonoids are a very

large subclass of polyphenolic derivatives widely present in

many plant sources. Many studies have reported that flavo-

noids possess the anticancer action with little side effect since

they can interfere with the progression of cancer through the

regulation of cell proliferation, apoptosis, invasion and metas-

tasis (16). Sen et al. (17) reported that epigallocatechin-3-gal-

late has the ability to inhibit MMP-2 expression in human

breast cancer cell line MCF-7. In addition, it is reported that

the genistein can inhibit the invasive potential of the hepato-

cellular cell Bel-7402 by changing the cell cycle, apoptosis,

and angiogenesis (18). Therefore, flavonoids may be a promis-

ing research direction for treating the tumors.

In this study, liquiritigenin (LQ; Fig. 1 A) is a type of flavo-

noids extracted from conventional herbal medicine – glycyrrhi-

zae. Evidence suggests that LQ possesses the antitumor effects

in 5 human cancer cell lines in vitro (19). The previous research

has documented that LQ also has the antiangiogenic effect by

inhibiting HIF-1 a and VEGF expression through the AKT/

mTORp70S6 K signaling pathway in human cervical carci-

noma HELA cells (20). Furthermore, LQ could inhibit the

migration of lung cancer cell (A549) in a dose- and time-depen-

dent manner by monitoring the regulation of MMP-2 expres-

sion (21). Based on the above research, we supposed that LQ

has strong cancer suppressive effects in human cancers. How-

ever, it has not been explored that whether LQ combined with

the chemotherapy drugs can enhance the chemotherapy drugs

to produce a better inhibition effect on cells invasion and metas-

tasis, reducing toxicity of chemotherapy drugs. Therefore, in

this report, we try to study the effect and the molecule mecha-

nism of LQ combined with low dose chemotherapeutic drug

(CDDP) on the migration and invasion potential of the B16F10

melanoma cells in vitro and in vivo.

MATERIAL AND METHODS

Chemical and Regents

LQ (purity �99.68%, HPLC) and CDDP were respectively

purchased from Nanjing University of Chinese Medicine and

Nanjing First Hospital. Rabbit monoclonal antibodies against

phosphor-/total form of AKT and PI3 K, PI3 K/AKT inhibi-

tor-LY294002, the horseradish peroxidase (HRP)-conjugated

antirabbit IgG and antimouse IgG were purchased from Cell

Signaling Technology (Beverly, MA). MMP-2, MMP-9 anti-

bodies, the HRP-conjugated antigoat IgG were obtained from

Santa Cruz Biotechnology (Santa Cruz, CA). The b-actin

antibody was obtained from Boster Biological Technology

(Boster Biological Technology).

FIG. 1. Effect of liquiritigenin (LQ) combined with cis-diamine dichloroplati-

num (CDDP) on the proliferation of B16F10 cells. A: Chemical structure of

LQ. B: Effects of LQ individually on the cell proliferation in B16F10 cells.

The cells were treated with the different concentrations of LQ for 24 and 48 h,

the number of survived cells after each treatment was determined by MTT. C:

The cells were treated with LQ and CDDP alone or in combination for 24 and

48 h. Cell viability was determined by MTT assay and the values were

expressed as means § SD. Results are representative of 3 independent

experiments. *P < 0.05, **P < 0.01 compared with low-dose CDDP group;#P < 0.05, ##P < 0.01 compared with high-dose CDDP group; using one-way

analysis of variance.

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Cell Culture and Drug Treatment

The melanoma B16F10 cell line was kindly provided

by Nanjing University of Chinese Medicine. The cells were

cultured in RPMI 1640 medium supplemented with 10% fetal

bovine serum (FBS), penicillin (100 U/ml), and streptomycin

(100 mg/ml) at 37�C in 5% CO2 humidified atmosphere.

These cell culture reagents were all purchased from GIBCO

Life Technology (Grand Island, NY).

In vitro, LQ was dissolved in dimethyl sulphoxide

(DMSO; Sigma Chemicals Co., St. Louis, MO) and diluted

with RPMI 1640 to appropriate concentrations just before

use. In vivo, LQ was prepapred in 5% sodium carboxymeth-

ylcellulose. During the experiments, cells were treated with

DMSO at a final concentration of 0.2%. CDDP was dissolved

in double distilled water at a concentration of 0.5 mM as

reserve solubility.

Cell Viability Assay

Cell viability was assessed by methyl thiazolyl tetrazolium

(MTT) assay. B16F10 cells (3000 cells/well) were seeded in

96-well plates. In logarithmic growth period, cells were treated

with different doses of LQ (0, 25, 50, 100, 200 mM), or CDDP

(0, 2, 10 mM), or LQ combined with CDDP for 24 h and 48 h.

Then, MTT reagent (20 ul, 5 mg/ml in PBS) was added to

every well and the plates were further incubated at 37�C for 4

h. After the incubation, the medium was removed, and

replaced with 150 ul DMSO. Absorbance was measured at

570 nm by an automatic micro-plate reader (EL808, Bio-Tek,

Winooski, VT).

Wound-Healing Assay

Cells were cultured in 24-well plate at a density of 5 £ 104

cells/well with complete media, and then they were serum

starved overnight. Cell monolayer was straightly scratched

with a pipette tip, and the detached cells were removed by

washing with PBS for 2 times. Finally, cells were treated with

LQ or CDDP for 24 h and photographed.

Migration and Invasion Assays

The migration and invasion potential of cells were analyzed

by Boyden chamber assays using the upper chamber with

6.5-mm diameter polycarbonate filters (8 mM of pore size,

Corning Costar, Cambridge, MA). In addition, for the invasion

assay, the upper chambers should be coated with matrigel (50

ml/well, BD Biosciences, Franklin Lakes, NJ) and dried before

use. Cells were pretreated with the indicated doses of LQ and

CDDP for 24 h, then 100 ul of the pretreatment of B16F10

cells (3 £ 105cells/ml in serum-free medium) were added in

suspension to the upper chambers, and medium (600 ml,

supplemented with 10% FBS) was added to the lower cham-

bers. The filter insert was placed into the lower chamber and

incubate at 37�C for 24 h. After 24 h incubation, cells in the

upper chamber were carefully swabbed with cotton. Cells that

had penetrated the membrane were fixed in methanol, stained

with crystal violet, and photographed. Finally, the crystal vio-

let retained on the filters dissolved in 30% acetic acid, and the

absorbance was measured at 595 nm using a micro-plate

reader.

Gelatin Zymography

The MMP-2 and MMP-9 activity was examined by gelatin

zymography. The B16F10 cells were plated in 6-well plate.

Logarithmic phase of tumor cells were washed with serum-

free RPMI1640 medium 3 times, then serum-free RPMI1640

medium was added to each well with the indicated doses of

LQ and CDDP for 24 h. The conditioned medium was then

collected to the samples with loading buffer. The medium was

separated by electrophoresis on 10% sodium dodecyl sulfate

polyacrylamide gel electrophoresis containing 0.1% gelatin

and then the gels were soaked in 2.5% Triton X-100 in

dH2O twice for a total of 60 min at room temperature and

incubated in substrate buffer (50 mmol/l of Tris–HCl, 5mmol/l

of CaCl2, 0.02% NaN3 and 1% triton X-100, pH 8.0) at

37�C for 48 h. After these, the gels were stained with 0.1%

Coomassie Brilliant Blue R250 for 3 h, followed by destaining

with 10% acetic acid and 30% methanol. The gelatinolytic

activity was detected as clear bands against the blue

background.

Western Blotting

Cells were harvested, lysed, and centrifuged for 30 min at

12000 g at 4�C. The supernatants were needed, and the protein

concentrations were determined using a BCA protein assay kit

(Bio-Rad Labs, Hercules, CA). A total of 60 mg protein was

electrophoretically separated on a 6–12% SDS-polyacrylamide

gel and electroblotted onto polyvinylidence fluoride mem-

brane. After blocked with 5% nonfat milk in Tris buffered

saline containing 0.1% Tween (TBST) for 1-2 h, the immuno-

blots were probed with antibodies to b-Actin (1:1000), MMP-

2 (1:500), MMP-9 (1:1000), p-AKT (1:1000), and PI3 K

(1:1000) at 4�C overnight. Then the membranes were washed

with TBST for 30 min and incubated with second antibodies

as follows: HRP-conjugated goat antirabbit IgG (1:2000) or

HRP-conjugated goat anti-mouse IgG (1:2000) at room tem-

perature for 1 h. Reactive proteins were visualized with an

enhanced chemiluminescence detection kit (Cell Signaling

Technology, Beverly, MA). Each bolt was repeated 3 times.

Animals

Female C57 BL/6 black mice, 6–8 weeks old, were pur-

chased from Shanghai SLAC Laboratory Animal Co. Ltd

(Shanghai, China). All mice were kept in a steady environment

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at (23 § 2�C) with a 12:12 light–dark cycle. All the experi-

ments with animals proceed in accordance with the guidelines

of the institutional animal ethical committee.

Experimental Lung Metastasis Assay

B16F10 (2 £ 105 cells in 0.2 ml) were injected into the tail

vein of C57 BL/6 mice. Then the mice were divided into 7

groups (N D 8-10 for each group) as follows: Group A, blank

group; Group B, the control group that received saline of com-

parable volume to the drug; Group C and D, supplementation

respectively with a low-dose CDDP (1.5 mg/kg), a high-dose

CDDP (3 mg/kg); Group E, F, G, the combination groups and

treatment respectively with low-dose LQ and low-dose CDDP

(5 mg¢kg¡1¢d¡1 C 1.5 mg/kg), mid-dose LQ, and low-dose

CDDP (10 mg¢kg¡1¢d¡1 C 1.5 mg/ kg), high-dose LQ and

low-dose CDDP (20 mg¢kg¡1¢d¡1 C 1.5 mg/ kg). LQ was

administered intragastrically once per day and CDDP was

administered by intraperitoneal injection once every 3 days.

Mice in each group were weighed every other day. After

21 days, mice were killed. The lungs were removed and a

small part (about 0.01 g lung tissue from inferior lobe of left

lung) was fixed in 4% paraformaldehyde solution, others were

stored at -80�C, and used for the various experiments as

needed.

ELISA Measurements

Blood was sampled from mouse eyes and centrifuged to

obtain serum. The concentrations of MMP-2 and MMP-9 were

measured using commercially available enzyme linked immu-

nosorbent assay (ELISA) kits (R&D Systems, Minneapolis,

MN) according to the manufacturer’s instructions. Standard

curves were measured at the same time and used for calcula-

tion of the 2 MMPs levels in samples. The 2 MMPs levels

were expressed as ng/ml.

Histological Studies

The tissue pieces was disposed with paraffin method, 5-mM

sectioned, and subjected to hematoxylin and esosin. Finally,

the histopathological characteristics of the tissues were

observed and photographed under a microscope at magnifica-

tion 200 £ (Olympus, IX-70, Tokyo, Japan).

Statistical Analysis

All data are expressed as mean § SD from 3 independent

experiments and the differences were analyzed by Student’s

t-test or one-way analysis of variance (ANOVA). The differ-

ence was considered significant at levels of P < 0.05.

RESULTS

Effects of LQ Individually or in Combination with CDDPon the Viability in B16F10 Cells

Effects of LQ Individually on the Cell Viability

Cells were individually treated with LQ (25, 50, 100, and

200 mM) for 24 and 48 h. As shown in Fig. 1B, under the

concentration of 100 mM, LQ had no effect on cell viability

at 24 h (P ˃ 0.05). This result showed that LQ was not toxic

to B16F10 cells under 100 mM, so we used LQ to B16F10

cells under the concentration of 100 mM (25, 50, 100 mM)

for 24 h in following experiments.

The Enhancement Effect of LQ on CDDP Suppressing the

Viability in B16F10 Cells

To evaluate the effects of LQ and CDDP on the growth of

B16F10 cells, we detected the cell viability by MTT assay.

Fig. 1 C had showed that LQ (25, 50, 100, 200 mM) com-

bined with CDDP (2 mM) on B16F10 cells significantly

reduced cell viability in a time- and dose-dependent manner

compared to CDDP (2 mM)-treated only (P < 0.05). Besides

the combination of LQ at 100, 200 mM and CDDP (2 mM)

exerted stronger suppression effects on the cells viability than

CDDP (10 mM) –treated group (P < 0.05) (Fig. 1 C).These

results suggested that LQ can enhance the inhibitory effect of

CDDP on the viability in B16F10 cells.

The Enhancement Effect of LQ on CDDP SuppressingMigration and Invasion in B16F10 Cells

Effects on the Migration in B16F10 Cells

As shown in Fig. 2 A, the results of the Boyden chamber

assays also showed that LQ combined with CDDP (2 mM)

significantly suppressed cell migration (21.5%, 49.6%,

75.6%) in a concentration-dependent manner compared with

CDDP (2 mM)-treated alone (11%) (P < 0.05). Moreover,

the inhibition ability of LQ at 50 mM combined with CDDP

at 2 mM group was almost consistent with the CDDP (10

mM)-treated group (51.6%). These data indicated that LQ/

CDDP combinations could inhibit the migration of B16F10

cells, and to some degree, LQ could increase the inhibition

ability of CDDP on the cell migration.

Effects on the Invasion in B16F10 Cells

Apart from the migration ability, the cell invasive power is

also an important characteristic of tumor metastasis. Therefore,

we tested the inhibition effect of LQ combined with CDDP on

the invasion of B16F10 cells. As presented in Fig. 2B, LQ com-

bined with CDDP could result in dose-dependent decrease in

cell invasion after 24-h incubation. The inhibition rate of LQ

(50, 100 mM) combined with CDDP (2 mM) reached 51.4%

and 69.5%, whereas the CDDP (2 mM) treatment group is only

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up to 16.7%. Strikingly, the antiinvasion effect of LQ (100mM)

combined with CDDP group was stronger than the CDDP (10

mM)-treated group (54.4%). To sum up, LQ/CDDP combina-

tions exert a more remarkable inhibition action on the B16F10

cell invasion than the CDDP individual treatment groups, sug-

gesting that LQ could strength the effect of CDDP to inhibit

cell invasion.

The Enhancement Effect of LQ on CDDP SuppressingMigration and Invasion by Regulating the Activity andProtein Expression of MMP-2 and -9 and the PI3 K/AKTSignaling Pathway

Effects on the Activity and Protein Expression of MMP-2 and

-9 in B16F10 Cells

There is a general consensus that the MMP family is closely

related to cell migration and invasion. From the above results,

we found that LQ combined with CDDP had a remarkable

inhibition effect on B16F10 cells migration and invasion, this

enlightened us to study the effects on the expression of MMP-

2 and MMP-9. We examined the gelatinolytic activity of

MMP-2 and MMP-9 secreted from B16F10 cells. After that,

the protein expression of MMP-2 and MMP-9 were determi-

nate by Western blotting. As shown in Fig. 3 A and 3B, quanti-

fication analysis of the gelatinolytic activity data suggested that

MMP-2 activity reduced by 37.8%, 64.8%, and 72.3%, and

MMP-9 activity decreased by 23.7%, 59%, and 72.3% when

cells were disposed with the co-incubation of LQ with CDDP.

Moreover, the data demonstrated the co-incubation of LQ with

CDDP for 24 h significantly decreased the protein expression

of MMP-2 and MMP-9 compared with CDDP (2 mM)-treated

alone (P < 0.05). Treatment with LQ (50 mM) combined with

CDDP (2 mM) for 24 h significantly downregulated MMP-2

and MMP-9 expression approximately twofolds compared to

that of CDDP (2 mM) only. What’s more, the inhibition action

of CDDP (10 mM)-treated group on MMP-2 and MMP-9 was

lower than that of LQ (100 mM) combined with CDDP (2 mM)

group (P< 0.01) (Fig. 3C and 3D). From the above results, we

speculated that LQ raised the inhibitory effect of CDDP on the

invasion of B16F10 cells through downregulating the activity

and protein expression of MMP-2 and MMP-9.

Effects on the Protein Expression of PI3 K, PTEN, and p-AKT

in B16F10 cells

To further investigate whether the PI3 K pathway, take

effect in LQ combined with CDDP regulation of melanoma

migration and invasion, we first examined the expression

of PI3 K, p-AKT, and PTEN in B16F10 cells treated with

CDDP individually or combined with LQ for 24 h by

Western blotting. Results showed that either the high-

CDDP treated group or CDDP (2 mM) combined with LQ

group significantly suppressed AKT phosphorylation and

PI3 K expression and upregulated the expression of PTEN

compared with the CDDP (2 mM)-treated group (P <

0.05). However, when cells were only treated with CDDP

(2 mM), there was little effect (Fig. 4A).

Secondly, to verify LQ combined with CDDP may regu-

late MMP-2 and MMP-9 through the PI3 K/AKT pathway,

FIG. 2. Effect of liquiritigenin (LQ) combined with cis-diamine dichloroplatinum (CDDP) on the migration and invasion of B16F10 cells. A: The cell migration

ability was measured by the Boyden chamber assays. In the Boyden chamber assays, after cells were cultured in different doses of LQ and CDDP for 24 h, the

same number of cells were seeded in the upside of Transwell, after 24 h, the downward side of the membrane was stained with hematoxylin and eosin. Image

magnification: 100£. B: The cell invasion ability was also measured by the Boyden chamber assay, the only different event is the upside chamber coated with

matrigel. Besides, a: control; b: CDDP (2 mM); c: CDDP (10 mM); d: LQ (25 mM) C CDDP (2 mM); e: LQ (50 mM) C CDDP (2 mM); f: LQ (100 mM) CCDDP (2 mM). Each bar shows the mean § SD. Results are representative of three independent experiments. *P < 0.05 and **P< 0.01 compared with low-

dose CDDP group; #P < 0.05 and ##P < 0.01 compared with high-dose CDDP group; using one-way analysis of variance.

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we pretreated the B16F10 cells with LY294002, a PI3 K

inhibitor for 1 h and then incubated in the presence or

absence of LQ (50 mM) or CDDP (2 mM) for 24 h. Cell

lysis buffer was subjected to Western blotting to examine

the expression of MMP-2 and -9 and p-AKT. Fig. 4B

showed that LY294002 (20 mM) decreased the AKT phos-

phorylation and MMP-2 and -9 expression compared to the

control and mimicked the effect of LQ (50 mM) combined

with CDDP (2 mM) on these factors, whereas there was a

little change on cells treated with only LQ or CDDP.

What’s more, the expression of inactive AKT also sup-

pressed the abilities of invasion and migration in B16F10 cells

which was originally inhibited by LQ combined with CDDP

as analyzed by the wound-healing assay and Boyden chamber

assay (Fig. 4 C). Taken together, these results suggested that

LQ mediated its inhibition effect of CDDP on anti-

invasiveness in B16F10 cells mainly via down-regulation of

MMP-2/9 expression and inhibition of PI3 K signaling

pathway.

The Enhancement Effect of LQ on CDDP SuppressingMigration and Invasion in Experimental PulmonaryMetastasis Model

Effects on Mice Weight, Lung Weight, and Lung Index

To assess whether the enhancement effect of LQ on CDDP

suppressing migration and invasion in B16F10 cells can be

reproduced in vivo, we injected B16F10 cells into the tail vein

of mice and built the lung metastasis model. Table 1 showed

the changes in mice body weight, lung weight, and lung index

during the experiment. Data showed that the final bodies

FIG. 3. Effects of liquiritigenin (LQ) in combination with cis-diamine dichloroplatinum (CDDP) on the activity and protein expression of matrix metalloprotei-

nase (MMP)-2 and -9. After treatment of B16F10 cells with LQ combined with CDDP for 24 h, MMP-2 and -9 was analyzed by the gelatin zymography assay

and the Western blot. Intensity of each band was quantified by densitometry (Image J). The gelatinolytic activity and the protein expression of control group

were designated as 100%, and others were represented as folds compared with the control. Each bar shows the mean § SD. Results are representative of 3 inde-

pendent experiments. *P < 0.05 and **P< 0.01 compared with low-dose CDDP group; #P < 0.05 and ##P < 0.01 compared with high-dose CDDP group.

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weight of CDDP (L: 1.5 mg/kg) combined with LQ (L:

5 mg/kg, M: 10 mg/kg, H: 20 mg/kg) group were increased with

no significant difference compared to the initial body weight and

the tumor control group, suggesting that the concentrations of

LQ and CDDP in our study were within the safe scope. Mean-

while, the drug-treated groups except CDDP (L)-treated group

can significantly decreased weight of lung and the lung index

compared with the tumor control group (P< 0.05).

TABLE 1

Changes of body weight, lung weight, and lung index in different groups (means § SD)

Body weight (g) Lung index

Group Initial Final Lung weight (g) Lung weight (mg)/body weight (g)

Blank Control 18.1 § 0.59 19.2 § 0.79 0.12 § 0.014 6.15 § 0.77

Control 18.3 § 0.81 18.7 § 0.69 1.13 § 0.25 34.6 § 19.9

CDDP(L) 18.6 § 1.24 18.1 § 2.19 0.94 § 0.27 30.5 § 11.4

CDDP(H) 18.8 § 1.35 17.0 § 1.74 0.32 § 0.11** 13.9 § 3.21**

LQ(L) C CDDP(L) 18.6 § 0.89 19.4 § 1.58 0.71 § 0.21*## 21.5 § 14.8*##

LQ(M) C CDDP(L) 18.6 § 0.99 19.1 § 1.42 0.4 § 0.03** 14.1 § 2.49**

LQ(H) C CDDP(L) 19.0 § 0.87 19.5 § 0.97 0.25 § 0.016**# 7.40 § 0.99**#

Blank group (n D 8): no treatment; control group (n D 8): that received saline of comparable volume to the drug; CDDP(L) (n D 8): a low-dose CDDP (1.5 mg/kg); CDDP(H) (nD 8): a high-dose CDDP (3 mg /kg); LQ(L)CCDDP(L) (n D 8): low-dose LQ combined with low-dose CDDP (5 mg¢kg¡1¢d¡1C1.5 mg/ kg);LQ(M)CCDDP(L) (n D 8): mid-dose LQ combined with low-dose CDDP (10 mg¢kg¡1¢d¡1 C 1.5 mg/ kg); LQ(H)CCDDP(L) (n D 8): high-dose LQ combinedwith low-dose CDDP (20 mg¢kg¡1¢d¡1C1.5 mg/ kg). Liquiritigenin (LQ) was administered intragastrically once per day; cis-diamine dichloroplatinum (CDDP)was administered by intraperitoneal injection once every 3 days. Results show the mean § SD (n D 8–10).*P < 0.05 and **P< 0.01 compared with low-dose CDDP group. #P < 0.05 and ##P < 0.01 compared with high-dose CDDP group.

FIG. 4. Effects of liquiritigenin (LQ) in combination with cis-diamine dichloroplatinum (CDDP) on PI3 K/AKT pathway. A: After treatment of B16F10 cells

with LQ combined with CDDP for 24 h, PI3 K, p-AKT, and PTEN were determined by Western blot. B: Effect of PI3 K inhibitor (LY294002) on the protein

expression of p-AKT, matrix metalloproteinase (MMP)-2 and MMP-9. Cells were treated with LY294002 (20 mM) for 1 h and then incubated in the presence or

absence of LQ (50 mM) or CDDP (2 mM) for 24 h. C: Effect of PI3 K inhibitor (LY294002) on the cells migration and invasion. Intensity of each band was quan-

tified by densitometry (Image J). The protein expression of control group was designated as 100%, and others were represented as folds compared with the con-

trol. Each bar shows the mean § SD. Results are representative of 3 independent experiments. *P < 0.05 and **P< 0.01 compared with low-dose CDDP group;#P < 0.05 and ##P < 0.01 compared with high-dose CDDP group.

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Effects on the Lung Metastasis of Tumor Cells and

Histological Assessment

Figure 5 A showed that CDDP (L) combined with LQ (M,

H) groups and the high-CDDP treated group inhibited the

numbers of metastatic nodules compared to the tumor control

group. Whereas, the inhibition of the low-CDDP treated group

was not obvious. These results were further verified by the

hematoxylin-eosin staining performed to the lung tissue, the

lung metastatic nodules of tumor control group were larger

than that of the LQ/CDDP combination group and high-

CDDP treat group (Fig. 5 A).These results suggested that LQ

can enhanced the anti-invasive action of CDDP in vivo.

Effects on the Concentration of MMP-2 and -9 in Serum

In clinic, many researches had reported that the content of

MMP-2 and -9 in serum had certain relevance with invasion

and metastasis of tumor (22), so we detected the concentra-

tion of MMP – 2/9 in the mice serum with ELISA method.

As shown in Fig. 5 C and 5 D, LQ (L, M, H) combined with

CDDP (L) significantly reduced the concentration of MMP-2

and -9 in a dose-independence method compared to the tumor

control group (P < 0.05). Simultaneously, the content of

MMP-2 and -9 in LQ (M) combined with CDDP (L) group

was almost equal to that of the high-CDDP treat group.

Effects on the Protein Expression of PI3 K, p-AKT, and PTEN

in Lung Tissues

The experimental results in vivo were consistent with our in

vitro result. Fig. 5B showed that combination of LQ and

CDDP could more significantly inhibit PI3 K and p-AKT

expression level and upregulate PTEN expression level than

CDDP treatment alone at a low dose.

Taken together, LQ in combination with CDDP could exert

a significant inhibition on experimental pulmonary metastasis

of tumor in vivo compared to the CDDP treatment alone.

DISCUSSION

Currently, CDDP has been proved to be one of the second-

line drugs for patients with metastatic melanoma, but the side

effects of the therapy provide a short-lived palliative care (23).

Thus, a growing number of research have been focused on

whether antineoplastic components of pure plants can enhance

the anti-tumor and anti-metastatic activity action of low-dose

FIG. 5. Effects of liquiritigenin (LQ) combined with cis-diamine dichloroplatinum (CDDP) on the lung metastasis of B16F10 cells in C57BL6 mice. A: The

model of experimental pulmonary metastasis of B16F10 cells and the pathological analysis of lung tissue of different treatment groups. B: Effects of LQ in com-

bination with CDDP on the protein expression of PI3 K, p-AKT, and PTEN in lung tissues. CDDP(L) and CDDP(H) are represented CDDP at 2 mM and 10 mM;

LQ(L), LQ(M), and LQ(H) stand for LQ at 25, 50, and 100, mM respectively. C,D: Effects of LQ in combination with CDDP on the concentration of matrix met-

alloproteinase (MMP)-2 and -9 in mice serum. Blood was sampled from mice eyes and centrifuged to obtain serum. The concentrations of MMP-2 and MMP-9

were measured using commercially available enzyme linked immunosorbent assay (ELISA). Results show the mean § SD (n D 8–10). *P < 0.05 and **P<

0.01 compared with low-dose CDDP group, #P < 0.05 and ##P < 0.01 compared with high-dose CDDP group.

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chemotherapy drugs (24,25). In our study, LQ is a sort of flavo-

noids that have been reported on their antitumor properties.

Moreover, CDDP combined with retinoic acid reduced the

number of lung metastatic nodules of B16F10 cells in vivo (26).

Thus, we further investigated whether LQ combined with

CDDP may enhance the antiinvasion effect of CDDP in the

B16F10 melanoma cells and in experimental pulmonary metas-

tasis model. Firstly, our results showed that LQ combined with

CDDP could inhibit the growth of B16F10 cells in a dose-

dependent manner compared to the low-dose CDDP treatment

alone. Secondly, it is noteworthy that combinations of LQ and

CDDP had a stronger inhibitory effect on the migration and

invasion of B16F10 cells than CDDP alone at the same concen-

tration. Moreover, LQ–CDDP combination at a high dose had

a better suppression effect on the migration and invasion of

B16F10 cells than CDDP used individually at a high

concentration.

Our in vivo study is in accordance with the result of in vitro

study. CDDP alone cannot reduce the numbers of lung meta-

static nodules at a low dose. However, when LQ is added, the

lung metastasis of B16F10 cells were obviously inhibited in a

dose-dependent patter. Importantly, the combination of LQ and

CDDP did not cause the body toxicity because the mice

weights being treated with the combination of drugs were close

to the control mice. Therefore, our studies have demonstrated

that LQ may potentiate the antitumor invasion and metastasis

effect of CDDP in vitro and in vivo, suggesting that maybe a

significant advance in chemotherapy treating melanoma.

MMP-2 and -9, which can degrade Type IV collagen, an

integral part of the basement membrane, has demonstrated as

key regulators of cancer cell invasion and metastasis (27,28).

In melanoma, quercetin inhibits the invasion of murine mela-

noma B16-BL6 cells by decreasing pro-MMP-9 (29). Lu et al.

has reported that oroxylin A, one of the main bioactive flavo-

noid extracted from Scutellaria radix, inhibited the human

breast carcinoma cell MDA-MB-231 invasion and migration

through reduction of the activity and expression levels of

MMP-2 and MMP-9 (30). Thus, in our study, we had mea-

sured the expression of gelatinase (MMP-2 and -9) by Western

blotting analysis. Our studies had showed that LQ and CDDP

combination inhibited the expression of MMP-2 and MMP-9

in a dose-dependent manner and had a more potent suppres-

sion effect than CDDP alone in B16F10 cells. In addition,

CDDP at a high-dose of 10 mM could exert the inhibition

effect on the expression of MMP-2 and MMP-9. Previous

study had showed that CDDP had the potential for inhibition

on the invasion and metastasis in some cell lines, such as

HELA, A549 cells through regulating the MMPs system (31).

What’s more, in clinical trials, serum level of MMP-2 and -9

is a useful marker for the evaluation of diagnosis and progno-

sis in patients with cancer (32,33). Therefore, we tested the

serum content of MMP-2 and -9 in mice and the data showed

that the high-dose CDDP and the combination of LQ/CDDP

significantly reduced the content of MMP-2 and -9 compared

to the low-dose CDDP treatment. Based on these findings, it is

possible that the anti-invasion and metastasis activity of LQ

combined with CDDP could at least in part be owe to the

capacity to downregulate MMP-2 and MMP-9 in vitro and in

vivo.

Multiple genetic changes occur during the process of tumor

metastasis. Recognizing key proteins, such as PI3 K, AKT,

and PTEN referred to this process is necessary for understand-

ing tumor metastasis and researching new therapies. Several

studies have established that PI3 K/AKT pathway plays a cru-

cial role in cancer progression, including growth, transforma-

tion motility, and invasion (34,35). Abnormal PI3 K signaling

is a high occurrence in melanoma (36). Constitutive activation

of AKT and low expression of PTEN, a negative regulator of

PI3 K/AKT signaling, have been verified in relation to promot-

ing the tumor progression (37,38). Shin et al. has showed that

the inhibitory effects of anthocyanins on motility and invasion

of HCT-116 cells were associated with the PI3 K/AKT path-

way (39). In the present study, we found that the combination

of LQ and CDDP significantly suppressed the protein expres-

sion of PI3 K and AKT, upregulated the PTEN levels com-

pared to the low-dose CDDP in vitro and in vivo. Moreover,

the treatment of the melanoma cells with LY294002, a PI3 K

inhibition, resulted in a reduction in cell migration and inva-

sion, blocking AKT phosphorylation, and suppressing MMP-2

and -9 protein levels. Amer et al. had demonstrated that cis-

platin and PI3 K inhibition (LY294002) decreased invasion

and migration of human ovarian carcinoma cells via downre-

gulation of activated MMP-2 (40). Based on these above anal-

yses, it is possible that LQ enhanced the suppression of CDDP

on cell invasion and metastasis by regulating the MMP-2 and

-9 expression and the inhibition of activated PI3 K /PTEN/

AKT pathway in vitro and in vivo.

CONCLUSIONS

In conclusion, in this study, we show evidence that LQ

plays an intensive role on CDDP inhibiting the B16F10 cells

migration and invasion in vitro and in vivo. The possible

mechanism of inhibition effects is the PI3 K/AKT signaling

pathway including downregulation the expression of PI3 K,

AKT, upregulation the expression of PTEN, subsequently

decreasing MMP-2 and MMP-9 protein levels.

ACKNOWLEDGMENTS

We thank Yu-Chen Wu, Fei Jiang, Xue-Min Wang and Zhi-

Min Chen (Nanjing Medical University, China) for their tech-

nical assistance.

FUNDING

This work was supported by the National Natural Science

Foundation of China (No. 30972478) and the Priority

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Academic Program Development of Jiangsu Higher Education

institutions (China).

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