Anthocyanins from the Fruit of Have Anti-cancer Properties by Inhibiting in Cancer Proliferation, Invasion, Adhesion, and Angiogenesis in Breast Cancer Cells

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48 책임저자：이원섭

󰂕 660-702, 진주시 칠암동 90

경상대학교 의과대학 내과학교실, 건강과학원, 경남지역암센터

Tel: 055-750-8733, Fax: 055-758-9122

E-mail: lwshmo@hanmail.net or lwshmo@gshp.gsnu.ac.kr 정진명

󰂕 660-702, 진주시 칠암동 90

경상대학교 의과대학 신경외과학교실, 건강과학원, 경남지역암센터

Tel: 055-750-8110, Fax: 055-758-9122 E-mail: gnuhjjm@gnu.ac.kr

접수일：2013년 2월 18일, 1차 수정일：2013년 2월 22일, 2차 수정일：2013년 2월 26일, 게재승인일：2013년 2월 28일

Correspondence to：Won Sup Lee

Department of Internal Medicine, Institute of Health Sciences and Gyeongnam Regional Cancer Center, Gyeongsang National University School of Medicine, 90, Chilam-dong, Jinju 660-702, Korea

Tel: ＋82-55-750-8733, Fax: ＋82-55-758-9122 E-mail: lwshmo@hanmail.net or lwshmo@gshp.gsnu.ac.kr Co-corresponding author：Jin-Myung Jung

Department of Neurosurgery, Institute of Health Sciences and Gyeongnam Regional Cancer Center, Gyeongsang National University School of Medicine, 90, Chilam-dong, Jinju 660-702, Korea

Tel: ＋82-55-750-8110, Fax: ＋82-55-758-9122 E-mail: gnuhjjm@gsnu.ac.kr

Anthocyanins from the Fruit of Vitis coignetiae Pulliat Have Anti-cancer Properties by Inhibiting in Cancer Proliferation, Invasion, Adhesion, and Angiogenesis in

Breast Cancer Cells

Min Jeong Kim¹, Won Sup Lee¹, Jeong Won Yun¹, Jing Nan Lu¹, Ji Hyun Jung¹, Sang Mi Lee¹, Seok-Hyun Kim², Hye Jung Kim³, Jeong-Hee Lee⁴, Woon-Tae Jung¹, Yung Hyun Choi⁵, Gon-Sup Kim⁶, Jin-Myung Jung⁷,

Chung Ho Ryu⁸, Sung Chul Shin⁹ and Soon Chan Hong¹⁰

Departments of ¹Internal Medicine, ³Pharmacology, ⁴Pathology, ⁷Neurosurgery, and ¹⁰Surgery, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-702, ²Division of Hematology and Medical Oncology, Department of Internal Medicine, Sungkyunkwan University, Changwon 630-723, ⁶School of Veterinary Medicine Research

Institute of Life Science, and ⁹Department of Chemistry, Gyeongsang National University, Jinju 660-701,

5Department of Biochemistry, Dong-Eui University College of Oriental Medicine and Department of Biomaterial Control (BK21 program), Dong-Eui University Graduate School, Busan 614-052, ⁸Division of Applied Life Science (BK 21

Program), Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Korea

Vitis coignetiae Pulliat (Meoru in Korea) has been used as a remedy for inflammatory diseases and various cancers. Although a previous study have shown that anthocyanins from fruits of Vitis coignetiae Pulliat (AIMs) have anti-NF-κB activities. It is still not known whether AIMs suppress NF-κB-regulated molecules in breast cancer cells. This study was to determine the molecules involved in AIM-induced anticancer effects especially focusing on cancer metastasis. We found that AIMs suppressed cancer cell proliferation, migration and invasion. This also suppressed the NF-κB-regulated proteins involved in caner proliferation, invasion, adhesion and angiogenesis. In conclusion, this study demonstrates that the anthocyanins isolated from fruits of Vitis coignetiae Pulliat inhibit the NF-κB downstream molecules involved in caner proliferation, invasion, adhesion and angiogenesis, implying that AIMs have anti- metastatic properties. (Cancer Prev Res 18, 48-54, 2013)

Key Words: Anthocyanins, Vitis coignetiae Pulliat, Proliferation, Invasion, Adhesion, Breast cancer

INTRODUCTION

Breast cancer is considered as systemic disease because ma-

jority of treatment failure after breast surgery is caused by the systemic dissemination of cancer cells rather than an inadequate surgery. Therefore, the control of the metastatic activity is cru- cial for the management of breast cancer. The conventional sys-

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temic chemotherapy is a mainstay of breast cancer treatment for the inoperable breast cancer patients, shown to prolong the survival and improve quality of life. However, the most of elderly patients have experienced serious side effects from chemotherapy. Some of these patients die of complications of chemotherapy.

As advances in medical science, our lifespan has been extended. The population of elderly cancer patients and their cancer-related mortality are expected to increase because the elderly have a high risk for cancer development. Therefore, it is essential to find out less toxic agents in controlling cancer for this population.

Recently dietary agents are known to safely modulate phys- iological function and enhance anti-cancer activity^1~4) and to down-regulate the NF-κB-regulated gene expression.^5,6) Vitis coignetiae Pulliat (Meoru in Korea) traditionally has been used in Korean folk medicine for the treatment of inflammatory lesions and cancer. It contains abundance of anthocyanins be- longing to a class of flavonoids. Recently, in vitro and in vivo anti-cancer activities of anthocyanins have been demonstrated regarding anti-angiognesis and cancer invasion.^7,8) We pre- viously suggested that the anthocyanins isolated from Vitis coignetiae Pulliat (AIMs) may suppress cancer invasion through suppression of NF-κB pathway.⁹⁾ However, few studies were conducted to determine whether AIMs inhibit the NF-κB- regulated proteins involved in cancer proliferation, invasion, adhesion and angiogenesis. It is important to know which molecules are involved in AIM-induced anti-cancer activities. Here, we determine whether NF-κB-regulated proteins are involved in AIM-induced anti-cancer activity.

MATERIALS AND METHODS 1. Cell culture and chemicals

Human breast cancer cell lines MCF-7 cells from the American type culture collection (Rockville, MD, USA) were cultured in RPMI 1640 medium (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA), 1 mM L-gluta- mine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37^oC in a humidified atmosphere of 95% air and 5% CO2. Caspase activity assay kits were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies against cyclin D1, c-Myc, MMP-2, ICAM-1, and VEGF were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA, USA). Antibody against β -actin was from Sigma (Beverly, MA, USA). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin, and an enhanced chemiluminescence (ECL) kit were purchased from Amersham (Arlington Heights, IL, USA). All other chemicals not specifically cited here were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anthocyanins were isolated from Vitis coignetiae Pulliat.

2. Cell proliferation assays

For the cell viability assay, the cells were seeded onto 24-well plates at a concentration of 5×10⁴ cells/ml, grown to 70% confluence and then treated with the indicated concentration of AIMs for 48 h. Control cells were supplemented with media containing 0.1% DMSO (vehicle control). Following treatment, cell viability was determined by MTT assays.

3. Wound healing assay

MCF-7 cells were grown on 35 mm dish plate to 100% con- fluent monolayer and then scratched to form a 100 μm scratching wound using sterile pipette tips. The cells were then cultured with and without AIMs (400 μg/ml) in serum-free media for 24 h. The results were photographed with an Olympus photomicroscope at 12 h and 24 h after scratching cancer cell on the plate.

4. Cell invasion assay

For the cell invasion assays, MCF-7 cells were cultured in serum-free media overnight. 5×10⁴ cells were loaded onto pre-coated Matrigel 24-well invasion chambers (BD Bioscien- ces, San Jose, CA, USA) with and without AIMs (400 μg/ml).

Then 0.5 ml of medium containing 20% FBS was added to the wells of the plate to serve as a chemoattractant. The Matrigel invasion chambers were incubated for 18 h. We re- moved the cells on the Matrigel. Invading cells were fixed with 10% formalin, stained with DAPI, and counted.

5. Gelatin zymography

The gelatinolytic activities for MMP-2 and MMP-9 in the culture medium were assayed by electrophoresis on 10% poly- acrylamide gels containing 1 mg/ml gelatin at 4^oC. Polyacry- lamide gels were run at 120 V, washed in 2.5% Triton X-100 for 1 h, and then incubated for 16 h at 37^oC in activation buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2). After stain-

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ing with Coomassie blue (10% glacial acetic acid, 30% methanol and 1.5% Coomassie brilliant blue) for 2∼3 h, the gel was washed with a solution of 10% glacial acetic acid and 30%

methanol without Coomassie blue for 1 h. White lysis zones indicating gelatin degradation were revealed by staining with Coomassie brilliant blue.

6. Western blotting

The procedure was performed as described previously.¹⁰⁾ In brief, the concentrations of cell lysate proteins were determined by means of the Bradford protein assay (Biorad Lab., Ricmond, CA, USA) using bovine serum albumin as the standard. The protein (30 μg) was resolved by electrophoresis, electrotrans- ferred to a polyvinylidene difluoride membrane (Millipore, Bed- ford, MA, USA), and then incubated with primary antibodies followed by secondary antibody conjugated to peroxidase. Blots were developed with an ECL detection system.

7. Statistics

Each experiment was performed in triplicate. The results were expressed as means±SD. Significant differences were determined using the Student's t-test. Statistical significance was defined as p＜0.05.

RESULTS

1. AIMs suppress the proliferation, migration, and invasion of MCF-7 cells

First, we assessed the effects of AIMs on the cell growth.

MTT assay revealed that AIMs suppressed the proliferation of the cells in a dose-dependent manner (Fig. 1A). AIMs inhibited cell proliferation by about 50% at a concentration of 400 μg/

ml compared with controls. AIMs inhibited cancer cell migration in the wound healing test at the concentration of 400 μg/

ml (Fig. 1B). AIMs also remarkably inhibited the invasion of MCF-7 cells at the concentration of 400 μg/ml as measured by Matrigel invasion assays (Fig. 1C, 1D). These results suggest that AIMs have anti-cancer properties on proliferation, migration and invasion.

2. AIMs suppress cyclin D1 and c-Myc involved in cancer proliferation

When the cancer cell reaches the circulatory system to make metastatic foci, their survival depends on the cell survival.

Cyclin D1 and c-Myc involved in cancer cell proliferation and overexpressed in a variety of cancers.^11,12) These genes are regulated by NF-κB. TNF-α is one of the most potent NF-κB stimulants and usually increased in the patients with advanced cancers.^13,14) In this study, we used TNF-α as a NF-κB stimulant to clearly demonstrate the relationship between the anti-cancer effects of AIMs and NF-κB-regulated proteins involved in cell proliferation (cyclin D1 and c-Myc). Western blotting revealed that TNF stimulated the expression of cyclin D1 and c-Myc, and that AIMs explicitly inhibited their expression (Fig. 2).

3. AIMs suppress secreted MMP-2 and MMP-9 proteins involved in cancer invasion

Tumor invasion is the first step for the metastasis. This process includes proteolytic digestion of the ECM, and cell migration through the basement membranes to reach the circulatory system.

Particularly, MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) are involved in the proteolytic digestion of the basement membrane by degrading type IV collagen, which are regulated by NF-κB.¹⁵⁾ Therefore, we assessed the effects of AIMs on MMP-2 and MMP-9 by gelatin zymographic analysis, because secreted MMP-2 and MMP-9 plays important roles in cancer invasion.

As indicated in Fig. 3, AIMs markedly suppressed the gelatinolytic activities of secreted MMP-2 and MMP-9 in dose-dependent manners.

4. AIMs suppress ICAM-1 and VEGF involved in cancer cell adhesion and angiogenesis, respectively

The roles of ICAM-1, and VEGF in adhesion and angiogenesis of cancer are well known. We also assessed the expression of ICAM-1, and VEGF. Here we also used TNF to clearly demonstrate the effects of AIMs on the expression of ICAM-1, and VEGF. The western blot revealed that TNF induced the expression of ICAM-1, and VEGF in a time-dependent manner and AIMs suppressed the TNF-induced expression of ICAM-1 and VEGF (Fig. 4).

5. AIMs inhibit IκBα phosphorylation induced by TNF-α

Mostly, NF-κB consists of a heterotrimer of p50, p65, and IκBα in the cytoplasm; when activated, the heterdimer of p50 and p65 is translocated into the nucleus in the sequence

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Fig. 1. The inhibitory effects of AIMs on cancer cell proliferation, migration and invasion in MCF-7 human breast cancer cells.

(A) Cell proliferation was assessed by MTT assay. The cells were treated with indicated concentrations of AIMs for 48 h. The data are shown as means±SD of three independent experiments. *p＜0.05 between the treated and the untreated control group.

(B) Cells were grown to 100% confluency on 30-mm cell culture dishes coated with rat tail collagen and then treated with or without AIMs (400 μg/ml) for 24 h. Photographs of the wounded area were taken at the interval of 0 h, 12 h, and 24 h after the scratch to evaluate cell movement into the wounded area. (C and D) The cells were exposed to AIMs for 6 h.

The cells (5×10⁴ cells) were loaded on pre-coated Matrigel 24-well invasion chambers (BD Biosciences) in the presence or absence of AIMs (400 μg/ml). Medium containing 20% FBS was placed in the basolateral chamber to act as a chemoattractant.

After 18 h, the cells on the apical side were wiped off using a Q-tip. Next, the cells on the bottom of the filter were stained with DAPI solution and then counted. The effects of AIMs are represented as percentages of the values of untreated control cells (% of control). *p＜0.05 versus control.

of events leading to activation. IκBα serves as an NF-κB inhibitor. Hence, the degradation of inhibitory κBα (IκBα) through phosphorylation by kinases is prerequisite to activate NF-κB. Here we confirmed whether AIMs suppressed TNF- α-induced phosphorylation of IκBα. The degradation of Iκ Bα through phosphorylation was seen as early as 5 min after adding TNF-α, and the pre-treatment with AIMs prevented the TNF-α-induced phosphorylation of IκBα in MCF-7 cells (Fig. 5).

DISCUSSION

NF-κB activation pathway is involved in the inflammation, cancer cell proliferation, invasion, adhesion, and angiogenesis.^16,17) Edible berry juice containing plentiful anthocyanins and dietary anthocyanidin delphinidin have been reported to involve NF-κ B pathway.^18,19) In addition, NF-κB is known to play significant roles in cancer cell invasion and metastasis.^16,17)

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Fig. 2. The inhibitory effects of AIMs on the expressions of cyclin D1 and c-Myc involved in cancer proliferation in MCF-7 cells. Cells were pretreated with AIMs (400 μg/ml) for 24 h and then treated with TNF-α (10 ng/ml) for 24 h. The expressions of cyclin D1 and c-Myc were assessed by Western blot analysis. Actin was used as the internal control.

Each result is a representative of at least two independent experiments.

Fig. 3. The inhibitory effects of AIMs on MMP-2 and MMP-9 involved in cancer invasion in MCF-7 cells. (A and B) MMP-2 and MMP-9 protein levels were measured by gelatin zymography. (C) Cells were pretreated with AIMs (400 μg/ml) for 24 h and then treated with TNF-α (10 ng/ml) for 24 hr. Actin was used as the internal control. Each result is a representative of at least two independent experiments.

This study was designed to investigate the effects of AIMs on NF-κB-regulated proteins and cellular responses stimulated by TNF-α in breast cancer cells. We found that AIMs suppress NF- κB activation induced by TNF-α in MCF-7 cells and the ex-

pression of NF-κB -regulated gene involved in cell proliferation (e.g., cyclin D1 and COX-2), anti-apoptosis (e.g., XIAP, IAP1, IAP2, Bcl-2, and Bcl-XL), and invasion & angiogenesis (e.g., MMP-2, MMP-9, ICAM-1, and VEGF). The NF-κB inhibition resulted at least in part from phosphorylation of IκBα. This report is the first to suggest that AIMs could suppress NF-κB activation and the NF-κB-regulated gene expression in breast cancer cells. The finding that AIMs have some anti-invasive effects by the suppression of MMP-2/ MMP-9 and NF-κB activation is consistent with the previous reports using anthocyanins from another source.^20,21)

In this study, we also suggested that AIMs may have anti-metastasis effects because AIMs suppressed the gene expression involving cell proliferation, anti-apoptosis, invasion, and angiogenesis which are all required to make metastatic lesions.

Tumor invasion is the first step for the metastasis. This process includes proteolytic digestion of the ECM, and cell migration through the basement membranes to reach the circulatory

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Fig. 4. The inhibitory effects of AIMs on the expressions of ICAM-1 and VEGF involved in cancer cell adhesion and angiogenesis, respectively in MCF-7 cells. Cells were pretreated with AIMs (400 μg/ml) for 24 h and then treated with TNF-α (10 ng/ml) for 24 h. The expressions of ICAM-1 and VEGF were assessed by Western blot analysis. Actin was used as the internal control. Each result is a representative of at least two independent experiments.

Fig. 5. Inhibitory effects of AIMs on IκBα phosphorylation.

Cells were pretreated with AIMs (400 μg/ml) for 24 h and then treated with TNF-α (10 ng/ml) for the indicated times. 1.

control, 2∼4. TNF-α alone group, 2 min, 5 min, 10 min after TNF-α treatment, 5∼7. AIMs＋TNF-α group, 2 min, 5 min, 10 min after TNF-α treatment. Data were representative of two independent experiments.

system. Particularly, MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) are involved in the proteolytic digestion of the basement membrane by degrading type IV collagen.¹⁵⁾ We demonstrated that AIMs had inhibitory activities by suppressing the expression of MMP-2 and MMP-9 in MCF-7 cells, which is consistent with our previous study demonstrating that AIMs had inhibitory activities on the expression of MMP-2 and MMP-9 in hepatocelluar carcinoma cells.^9,22)

When the cancer cell reaches the circulatory system to make metastatic foci, their survival depends on the anti-apoptotic apoptotic protein expression. We found that AIMs inhibited the anti-apoptotic proteins such as Bcl-2, XIAP, IAP-1, and IAP-2. This is consistent with previous studies showing that some natural compounds inhibiting NF-κB activation have inhibitory effects on these anti-apotpotic proteins.^5,6,18)

We found that AIMs inhibit the NF-κB activity through inhibition of IκBα phosphorylation induced by TNF-α. The limitation of this study is that the mechanisms by which AIMs manifest their effects on NF-κB pathway is not fully eluci- dated. A previous report suggested that delphinidin inhibits the expression of kinase phospho-IKK which is a upstream kinase of phospho-IκBα. The expression of kinase phospho-IKK is also regulated by many other signals such as PI3K-Akt pathway. Here, we used TNF-α, one of the NF-κB stimulants to determine the linkage between NF-κB activation and invasion. We also found that TNF-α stimulated the proliferation of other cancer cells, but not MCF-7cells (data not shown). This is the reason why TNF-α marginally augmented the invasion of MCF-7 cells. The pathophysiological relevance

of TNF-α-induced NF-κB activation is underscored by the observation that TNF-α is usually increased in patients with advanced cancers.^13,14) Data presented here indicated that AIMs in the concentration of 400 μg/ml had the anti-invasive effects on MCF-7 human breast cancer cells. The concentrations used in the present study are consistent with those in many other studies on the anti-tumor effect of anthocyanins on cultured cells.^18,23)

CONCLUSION

This study indicated that AIMs have anticancer effects by inhibiting the NF-κB activation and downstream proteins through inhibition of IκB Phosphorylation. This study pro- vides evidence that AIMs might have anticancer effects on human breast cancer.

ACKNOWLEDGEMENT

This study was supported by a grant of the National R&D Program for Cancer Control, Ministry for Health, Welfare &

Family Affairs, Republic of Korea (0820050), and in part a grant from the Korean Cancer Research Institute, Republic of Korea.

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