European Journal of Pharmaceutical Sciences 25 (2005) 5765

Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells
Kriengsak Lirdprapamongkol a, c , Hiroaki Sakurai a, b , Noritaka Kawasaki a , Min-Kyung Choo a , Yurika Saitoh a , Yasushi Aozuka a , Pattama Singhirunnusorn a , Somsak Ruchirawat d , Jisnuson Svasti c, e , Ikuo Saiki a, b,
b

Division of Pathogenic Biochemistry, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan The 21th Century COE Program, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan c Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand d Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok 10210, Thailand e Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand Received 10 June 2004; received in revised form 22 December 2004; accepted 24 January 2005 Available online 7 March 2005

Abstract Vanillin, a food avoring agent, has been reported to show anti-mutagenic activity and to inhibit chemical carcinogenesis. In this study, we examined the effect of vanillin on the growth and metastasis of 4T1 mammary adenocarcinoma cells in BALB/c mice. Mice orally administered with vanillin showed signicantly reduced numbers of lung metastasized colonies compared to controls. In vitro studies revealed that vanillin, at concentrations that were not cytotoxic, inhibited invasion and migration of cancer cells and inhibited enzymatic activity of MMP-9 secreted by the cancer cells. Vanillin also showed growth inhibitory effect towards cancer cells in vitro. However, vanillic acid, a major metabolic product of vanillin in human and rat, was not active in these in vitro activity assays. Our ndings suggest that vanillin has anti-metastatic potential by decreasing invasiveness of cancer cells. Since vanillin is generally regarded as safe, it may be of value in the development of anti-metastatic drugs for cancer treatment. 2005 Elsevier B.V. All rights reserved.
Keywords: Anti-metastatic; Migration; Matrix metalloproteinase; Breast cancer

1. Introduction Metastasis, the spread of cancer in the body, is a major cause of death in cancer patients. The metastatic process is a multi-step phenomenon, by which cells in the primary tumor invade surrounding tissue, penetrate into blood and lymphatic vessels, so that they can travel to distant sites via the circulatory system, and extravasate into the organ parenchyma, proliferating to form metastatic colonies at secondary sites. Cancer invasion takes place when the cancer cells respond and migrate towards gradients of stimuli such as growth fac

Corresponding author. Tel.: +81 76 434 7620; fax: +81 76 434 5058. E-mail address: byosei@ms.toyama-mpu.ac.jp (I. Saiki).

tors, and also requires proteolysis of basement membrane (BM) and extracellular matrix (ECM) proteins to create a path for migration. Matrix metalloproteinases (MMPs) are a group of enzymes responsible for the proteolysis of BM and ECM proteins, and the expression level of MMPs appears to correlate with the invasiveness of cancer cells (Woodhouse et al., 1997). Current chemotherapeutic treatments generally aim to use cytotoxic agents to kill proliferated cancer cells, but such treatment can also affect rapidly growing normal cells. Moreover, regrowth of tumors often occurs after treatment, and repeated cycles of treatment and regrowth can lead to a lethal outcome. A new approach in cancer treatment is cytostatic therapy, which aims to stabilize a non-pathologic state of the

0928-0987/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.01.015

58

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765

disease or delay the time of tumor regrowth, following cytotoxic chemotherapy. Cytostatic therapy requires long-term treatment using agents with little or no cytotoxic activity to retard invasion or proliferation of cancer cells (Kohn and Liotta, 1995). Vanillin, a food avoring agent, has been shown to inhibit mutagenesis induced by chemical and physical mutagens in various models (Keshava et al., 1998; Shaughnessy et al., 2001). It also showed chemopreventive effect in chemical carcinogenesis models in the rat (Tsuda et al., 1994; Akagi et al., 1995). Vanillin has been reported to have potential as an agent for treatment of sickle cell anemia disease (Abraham et al., 1991). More recently, reports showed that vanillin displayed antioxidant activity (Kumar et al., 2004), inhibited DNA-dependent protein kinase and enhanced sensitivity of cancer cells to cisplatin (Durant and Karran, 2003). Vanillin was given generally regarded as safe (GRAS) status by the Flavor and Extract Manufacturers Association (FEMA) and recognized as suitable for food use by the Food and Drug Administration (FDA) (Opdyke, 1977). Several oral chronic toxicity studies with rat were reported, in which high levels of vanillin were consumed for extensive periods without adverse effects (Opdyke, 1977; Kirwin and Galvin, 1993). Matched groups of male and female rats, fed with diets containing vanillin at 1000 ppm for 2728 weeks, 10,000 ppm for 16 weeks, or 5000, 10,000 and 20,000 ppm for 2 years resulted in no effect on growth or hematology, as well as causing no macroscopic or microscopic changes in the tissues (Hagan et al., 1967). In another study (Hake and Rowe, 1963), rats were maintained for 91 days on diets containing vanillin at 3000, 10,000 and 50,000 ppm (equivalent to about 150, 500 and 2500 mg/(kg day), respectively). Records of appearance, behavior, growth, mortality, terminal body and organ weights, terminal hematologic examination and histological studies, revealed no adverse effects when the diet contained 3000 ppm vanillin. Mild adverse effects followed ingestion of the 10,000 ppm diet, and at 50,000 ppm, growth was depressed and the liver, kidneys and spleen were enlarged. In this report, we studied the anti-metastatic activity of vanillin in a mouse model, and then examined the effects of vanillin on the metastasis-associated processes including invasion, migration, MMP secretion and its enzymatic activity in vitro.

were suspended in 0.5% (hydroxypropyl) methyl cellulose (HPMC), while the control group received 0.5% HPMC as vehicle. 2.2. Animals BALB/c mice (5-week-old female) were purchased from Japan SLC Inc. (Hamamatsu, Japan). They were maintained in the Laboratory for Animal Experiments, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, under laminar air-ow conditions. Food and water were freely available. This study was conducted in accordance with the standards established by the Guidelines for the Care and Use of Laboratory Animals of Toyama Medical and Pharmaceutical University. 2.3. Cell culture A spontaneous mammary adenocarcinoma cell line 4T1, was obtained from the American Type Culture Collection (ATCC) and cultured as monolayers in Dulbeccos Modied Eagles Medium (DMEM; GIBCO BRL, Life Technologies Inc., NY, USA), supplemented with l-glutamine and 10% heat-inactivated fetal bovine serum (FBS; CELLect Gold FBS; ICN Pharmaceuticals, Montreal, QC, Canada). The culture was maintained at 37 C in a humidied atmosphere of 5% CO2 . 2.4. In vivo experimental metastasis The 4T1 cell is a mouse mammary adenocarcinoma cell line expressing estrogen receptor (ER-positive), which when implanted to the mammary fat pad in female BALB/c mice, produces a large tumor mass within 1 month and causes metastases in the lungs (Michigami et al., 2002). Tamoxifen is an anti-estrogenic agent used as standard anticancer drug for oral treatment of patients with ER-positive tumors, but the effect of tamoxifen has not been reported in this tumor model. We therefore examined the effect of vanillin and tamoxifen in our animal experiments by oral administration, since this is the main route for receiving vanillin and tamoxifen in human. The 4T1 cells were harvested and resuspended in cold phosphate-buffer saline (PBS) to a nal concentration of 1 107 cells/ml. The cell suspensions (100 l/mouse) were injected into mammary fat pad of anesthesized mice. Six mice in each group received vanillin (100 mg/(kg day)) or tamoxifen (75 mg/(kg day)) or vehicle by oral administration 6 times a week, starting on day 1 after tumor implantation. After sacricing the mice on day 31, anti-tumor effect was evaluated by measuring the size of the primary tumor at the implantation site, and counting the number of metastasized tumor colonies on the surface of the lungs. The primary tumor size was measured using a caliper square along the longer axes (a) and shorter axes (b), and tumor volumes were calculated by following formula; tumor volume (mm3 ) = ab2 /2.

2. Materials and methods 2.1. Chemicals For in vitro experiments, vanillin, vanillic acid and tamoxifen citrate salt (SigmaAldrich Japan K.K., Tokyo, Japan) were dissolved in dimethyl sulfoxide (DMSO) and kept as stock solutions at 20 C. The nal concentration of DMSO was kept below 0.2% throughout the in vitro study. Control group was treated with 0.2% DMSO as vehicle in all in vitro experiments. For in vivo experiments, vanillin and tamoxifen

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765

59

2.5. Cell viability assay Viability of cells was assessed using a WST-1 Cell Counting Kit (Wako Pure Chemical Industries, Osaka, Japan). Briey, cell suspensions in DMEM containing 10% FBS were seeded into a 96-well plate (4 103 /100 l/well), and incubated at 37 C in a humidied atmosphere of 5% CO2 . After 24 h incubation, additional media (100 l) containing the test compounds at various concentrations were added to the wells and further incubated for 24 h. WST-1 solution (10 l) was added to each well at 2 h before the end of experiment. The absorbance at 450 nm was measured using a microplate reader. Cell viability was determined from the absorbance of soluble formazan dye generated by living cells. 2.6. Invasion and migration assays Invasion assay was carried out by using a membrane invasion culture system (MICS), which is a bioassay to determine the capability of cancer cells to digest ECM proteins (Matrigel) and migrate through a barrier (lter) (Welch et al., 1989). Cell motility, a necessary property of cancer invasion, was assessed by a migration assay, which determines only the capability of cells to migrate through the lter, when the Matrigel was not included. So invasion and migration assays were carried out in the same way and differed only in the membrane lter coating process. The invasion and migration assays were carried out using Transwell cell culture chambers (Corning Costar, MA, USA) attached with membrane lter (8.0 m pore size; Nucleopore, CA, USA) as previously described (Nakamura et al., 2003) with some modications. Briey, the lters used in both assays were pre-coated with 1 g of bronectin (Iwaki Glass, Tokyo, Japan) on the lower surface and dried under laminar-air ow on a clean bench. Only the lters used for invasion assay were additionally coated with 1 g of Matrigel (BD Bioscience, MA, USA) on the upper surface and dried in the same way. The precoated lters (Matrigel/bronectin-coated lters for invasion assay or bronectin-coated lters for migration assay) were washed extensively in PBS and dried before use. The 4T1 cells were harvested and resuspended (1 106 /ml) in DMEM containing 10% FBS and the test compounds at various concentrations. The cell suspensions were incubated at 37 C for 30 min in a water bath before being added into the upper compartment of the Transwell chamber (100 l/chamber). Media containing test compounds at the required concentration were added to lower compartment of the Transwell chambers (600 l/chamber). The culture was incubated for 24 h at 37 C in a humidied atmostphere of 5% CO2 . Then, the cells on the upper surface of lters were removed by wiping with a cotton swab, and the lters were xed with 30% methanol, followed by staining with 0.5% crystal violet in 20% methanol. The lters containing the stained cells were removed from the Transwell chambers and individually transferred to separate wells in a 96-well plate. The crystal violet dye retained on the lters was extracted with 30%

acetic acid, and the absorbance was measured at 595 nm using a microplate reader. The number of invaded or migrated cells was determined from the absorbance of crystal violet dye extracted from the lters. 2.7. Gelatin zymography and MMP inhibition assay MMPs in conditioned media of the 4T1 cells were detected by gelatin zymography in which the separated enzyme bands in gelatin-incorporated SDS-polyacrylamide gel were allowed to digest gelatin in the gel after electrophoresis. Since MMPs are also present in serum, serum-free media were used for cell culture when studying the secretion of MMPs. The 4T1 cells were cultured in a 24-well plate (5 104 /500 l/well) for 24 h, after which, the wells were washed twice with PBS and further incubated in serum-free media (DMEM containing 0.1% BSA, 400 l/well) containing vehicle or vanillin at various concentrations for 24 h. The conditioned media were collected, centrifuged to remove debris and supernatants were collected and kept at 20 C until used. Gelatin zymography was carried out according to a previous report (Lee et al., 2003), by electrophoresing aliquots of the supernatant on a 7.5% polyacrylamide gel containing 0.1% SDS and 0.1% gelatin at 4 C. After electrophoresis, the gel was washed twice with rinsing buffer (50 mM TrisHCl containing 2.5% Triton X-100, 5 mM CaCl2 , 1 M ZnCl2 , 0.05% NaN3 ) at room temperature for 1 h to remove SDS, and incubated for 24 h at 37 C with incubation buffer (50 mM TrisHCl containing 5 mM CaCl2 , 1 M ZnCl2 , 0.05% NaN3 ). The gel was stained with staining solution (0.1% Coomassie Brilliant Blue, 10% acetic acid, 10% isopropanol) and destained with 10% acetic acid, 10% isopropanol. Enzyme-digested regions were visualized as clear bands on a blue background, the enzymatic activity was quantied by Chemi Dos XRS System (Bio-rad). To examine effect of compounds on the secretion of MMPs, the 4T1 cells were cultured in a 24-well plate (5 104 /500 l/well) for 18 h, then the cells were treated with vehicle or vanillin at various concentrations for 6 h by adding media (50 l) with or without vanillin. After that, the wells were washed twice with PBS and incubated in serum-free media without vanillin (400 l/well) for collecting the secreted MMPs in vanillinfree conditioned media over a 24-h period. The MMP inhibition assay was carried out to whether vanillin has a direct inhibitory effect on the enzymatic activity of MMPs. The conditioned media prepared from the control well (vehicle treated well) was used as source of the enzyme, and loaded into the gelatin gel. After electrophoresis, the gel was cut into slices, corresponding to the lanes, put into different boxes and incubated in buffer with or without vanillin for 24 h at 37 C, before staining. 2.8. Growth arrest curve Cell suspensions of 4T1 cells in DMEM containing 10% FBS were seeded into a series of 6-well plates (4 104 /well),

60

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765

and incubated at 37 C in a humidied atmosphere of 5% CO2 . After 24 h, additional media containing vanillin or vehicle were added to the wells, with nal vanillin concentration of 4 mM. At indicated times, triplicate wells were trypsinized for determining cell number with a hemocytometer. At the end of experiment, cells were photographed by phase-contrast microscope-coupled digital camera (original magnication 50). 2.9. Statistical analysis Data are expressed as mean S.D., and analyzed by Students two-tailed t-test to determine the signicance of differences between groups. A p-value lower than 0.05 was considered to be signicant. In some cases, statistical signicance was also conrmed by Dunnetts test and the F-test.

3. Results 3.1. Effect of vanillin on primary tumor growth and metastasis The 4T1 cell is an ER-positive cell line, so the effect of vanillin on tumor growth and metastasis was studied in parallel with tamoxifen. Repeated oral administration of vanillin (100 mg/kg) to tumor-bearing mice for 1 month signicantly reduced the number of lung metastasized colonies as compared to the vehicle group, while tamoxifen treatment (75 mg/kg) did not reduce lung metastasis (Fig. 1A). However, vanillin or tamoxifen treatment did not show any statistically signicant effect on primary tumor growth at the implantation site or on the body weight of the animal (Fig. 1B and C). Statistical signicance of differences between the vehicle group and treatment groups were analyzed by Students t-test, and conrmed by Dunnetts test and the F-test. 3.2. Inhibition of invasion and migration of 4T1 cells by vanillin Vanillic acid has been reported to be a major metabolic product of vanillin in the rat (Kirwin and Galvin, 1993). Thus, to further study the anti-metastatic effect observed above, the effects of vanillin and vanillic acid on in vitro invasion were investigated. The 4T1 cells were cultured in Transwell chambers for 24 h, with various concentrations of vanillin, vanillic acid or tamoxifen. In the range of non-cytotoxic concentrations, vanillin showed signicant inhibitory effect on in vitro invasion at 0.52 mM, resulting in 4057% inhibition, while viability remained greater than 97% (Fig. 2A). However, vanillic acid did not inhibit invasion over the same concentration range (Fig. 2B). On the other hand, tamoxifen showed signicant inhibition of invasion at a concentration, which affected viability (12.5 M), resulting in 28% inhibition of invasion with 65% viability (Fig. 2C).

Fig. 1. Effect of vanillin and tamoxifen on lung metastasis (A), primary tumor growth (B) and body weight (C) of tumor-bearing mice implanted with 4T1 cells. After tumor implantation at mammary fat pad, the mice received vanillin (100 mg/(kg day)) or tamoxifen (75 mg/(kg day)) or vehicle by oral administration for 1 month. The number of metastasized colonies was determined after sacrice on day 31. The tumor size and body weight were measured at time intervals. Data are expressed as mean S.D. from six mice in each group. Signicant difference from vehicle group is shown by (** P < 0.01).

Since migration is a crucial step in the invasion process, the effect of vanillin on the migration of 4T1 cells was studied using the same concentrations as those used in the invasion assay. Vanillin showed signicant inhibition of migration, with 2435% inhibition being observed at 0.52 mM (Fig. 2D), which was lower than the extent of inhibition of invasion (4057%) obtained using the same concentrations. 3.3. Effect of vanillin on MMP secretion and enzymatic activity Since MMPs are a group of enzymes that play an important role in cancer invasion, the gelatin zymography technique was used to study the effect of vanillin on the secretion and enzymatic activity of MMPs in the concentration range that inhibited invasion. Although both MMP2 and MMP-9 are detectable by gelatin zymography, only

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765

61

Fig. 2. Effect of vanillin (A and D), vanillic acid (B) and tamoxifen (C) on 4T1 cell viability, invasion and migration. Viability of cells was assessed by using WST-1 Cell Counting Kit after the cells were incubated in media containing test compounds at indicated concentrations for 24 h as described in Section 2. Assays for invasion and migration of the cells were carried out by using Transwell chambers consisting of 8.0 m pore size lters, but only the lters used for the invasion assay were coated with Matrigel on the upper surface. The cells were allowed to invade or migrate in the presence of test compounds at indicated concentrations for 24 h, after which, the number of invaded or migrated cells were determined by a colorimetric method as described in Section 2. Control wells of all experiments were treated with 0.2% DMSO as vehicle. Three independent experiments were performed, but data show one representative experiment and are expressed as mean S.D. from triplicate wells in that experiment. Signicant differences from control are shown by (* P < 0.05) and (** P < 0.01).

the band corresponding to MMP-9 was detected in conditioned media of 4T1 cells, while no MMP-2 activity band was observed. Since vanillin was present throughout the invasion assay, the effect of vanillin on MMP-9 activity was studied by culturing 4T1 cells in serum-free media containing vanillin at various concentrations for 24 h, followed by determining the MMP activity in the serum-free conditioned media. Signicant reduction of MMP-9 activity in the conditioned media was observed at 2 mM vanillin, representing 44% reduction (Fig. 3A). Two additional experiments were performed to determine whether vanillin affected secretion or enzymatic activity of MMP-9. The effect of vanillin on MMP-9 secretion was studied by culturing 4T1 cells in culture media containing vanillin (0.54 mM) for 6 h, followed by incubating the cells in serum-free media without vanillin for 24 h. Vanillin showed no inhibition of MMP-9 secretion over the concentration range studied (Fig. 3B). The direct inhibition of vanillin on the enzymatic activity of MMP-9 was also studied. To obtain MMPs for study, 4T1 cells were cultured 24 h in medium containing 10% FBS without vanillin for 24 h, washed twice with PBS, and further incubated in serum-free media without vanillin for 24 h. MMPs from the conditioned medium were run in gelatin gels and then allowed to digest gelatin in incubation buffer containing vanillin (0.54 mM). As, can be seen, vanillin signicantly inhibited the enzymatic activity of MMP-9, with 4651% inhibition being observed at 24 mM (Fig. 3C).

3.4. Growth arrest effect of vanillin on 4T1 cells The effect of vanillin and vanillic acid on cell growth was studied. The number of cells was assessed by the cell viability assay, except the cells were seeded at 1 103 /well and incubated with test compounds for 3 days. After treatment, vanillin signicantly reduced the number of cells with IC50 of about 2.6 mM, but vanillic acid did not show the reduction effect over the same concentration range of 0.25 mM (Fig. 4A). The effect of vanillin on the growth rate of 4T1 cells was studied by the growth curve assay, carried out by counting cells in the culture plate at various time points. The growth of 4T1 cells was signicantly arrested by vanillin (4 mM), with a ve-fold decrease in cell number being observed as compared with controls at 72 h after exposure, at the time when control cells reached conuence (Fig. 4B). No morphological differences could be detected between vanillin treated cells and untreated cells throughout the experiments, as shown in the photographs taken at 72 h after exposure to vehicle or vanillin (Fig. 4C and D).

4. Discussion In this report, the anti-metastatic activity of vanillin was observed in an animal model, followed by investigation of the mechanisms involved by in vitro studies. The anti-metastatic

62

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765

Fig. 3. Effect of vanillin on enzymatic activity and secretion of MMP-9 by 4T1 cells. (A) Gelatin zymogram analysis of conditioned media obtained by culturing the cells in serum-free media containing vanillin (0.52 mM) for 24 h. (B) For detecting secreted MMP-9, the cells were incubated in cultured media containing vanillin (0.54 mM) for 6 h, followed by 24 h incubation in serum-free media without vanillin to collect secreted MMP-9. The conditioned media were analyzed by gelatin zymography. (C) Study of direct effect of vanillin on MMP enzymatic activity. Conditioned media from vehicle treated 4T1 cells were used as source of enzyme and run on a gelatin gel. MMP-9 in the gelatin gels was allowed to digest gelatin in incubation buffer containing vanillin (0.54 mM) for 24 h before staining with coomassie blue. Experiments A and B were performed twice, while experiment C was performed three times, in each case with similar results. Relative gelatinolytic activity of MMP-9 in each sample is expressed as the relative density of the band compared to control. Data are expressed as mean S.D. from the two (A and B) or three (C) independent experiments performed, and signicant differences from control are shown by (* P < 0.05) and (** P < 0.01). Representative zymograms at each vanillin concentration are shown above the bar graph indicating gelatinolytic activity.

activity of vanillin was demonstrated by the lower numbers of lung metastasized colonies in vanillin treated mice. Tamoxifen did not appear to retard tumor growth or metastasis in this study. A recent report showed that tamoxifen decreased tumor growth in a rat mammary carcinoma implanted tumor

model, without reducing the number of metastases in vivo when used as a single drug treatment by daily i.p. administration of 3 mg/(kg day) in Fischer 344 rat implanted with Mat B-III cells (Guo et al., 2002), but this study differs from ours in terms of cell line, animal species, dosage and route of administration. The concentration of tamoxifen required to produce 50% growth inhibition (IC50 ) of 4T1 cells in our study was 9 M (data not shown). The level of tamoxifen in the serum of mice has also been investigated, and ICR mice given oral doses of 200 mg/kg tamoxifen daily showed tamoxifen levels of 1.6 M in serum at day 7 (Robinson et al., 1991), which is lower than the IC50 observed for 4T1 cells. This may explain the lack of anti-tumor effect of tamoxifen in our mouse model. In the same study, the tamoxifen treated mice did not show signs of toxicity, such as loss of body weight or death, after continuous treatment for more than 7 days, consistent with our observations. The major metabolite of vanillin in human and rat is vanillic acid, and rats given an oral dose of 100 mg/kg vanillin yielded 47% of the dose as vanillic acid in 48 h (Kirwin and Galvin, 1993). Although there is no information on vanillin metabolism in the mouse, we decided to check the effectiveness of vanillic acid, as well as vanillin, on the in vitro invasion capability of 4T1 cells. To separate the anti-metastatic activity of a compound from its cytotoxic effect, the inhibition of invasiveness must be observed at non-cytotoxic concentrations of the compound (Welch et al., 1989). The results from in vitro invasion and migration assays of 4T1 cells indicate that vanillin is the active compound, as revealed by cytotoxicindependent inhibition of invasion and migration. Thus, even if vanillin is metabolized to vanillic acid in the mouse, the active compound in terms of inhibition on invasion is still likely to be vanillin. Cell migration is a complex process, involving several growth factors, which induce cell migration by binding to receptors on cell surface and stimulating downstream signaling pathways, resulting in cytoskeletal reorganization and stimulation of motility machinery of the cell (Anand-Apte and Zetter, 1997). This cellular process provides a variety of molecular targets for the development of therapeutic agents inhibiting cancer invasion and metastasis (Price and Thompson, 2002; Fenteany and Zhu, 2003). Aspirin, a small molecule which structurally related to vanillin, is an example compound that causes inhibition of invasiveness in various cancer cell lines with different mechanisms. Aspirin decreased invasiveness of hepatoma cells by inhibiting ERK1/2 activity in the downstream signaling pathway of HGF (Abiru et al., 2002). Invasion and migration of hepatoma cells were inhibited by aspirin via inhibiting MMP-2 secretion and increasing E-Cadherin production (Jiang et al., 2001). Aspirin inhibited migration of prostate cancer cells through actin cytoskeletal reorganization and suppressing constitutively active NF-B, resulting in inhibition of uPA-dependent migration (Lloyd et al., 2003). The mechanism of vanillin in inhibiting 4T1 cell migration may similar to one of the actions of aspirin or involve a different mechanism.

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765

63

Fig. 4. Effect of vanillin and vanillic acid on 4T1 cell growth. (A) For the cell growth assay, cells were incubated with various concentrations of test compounds (0.25 mM) for 3 days, after which the number of cells was assessed by the cell viability assay as described in Section 2. (B) Effect of vanillin on the growth rate was examined by growth curve assay, the cells were incubated with vehicle (0.2% DMSO) or 4 mM vanillin for 3 days, after which the cell numbers were counted by hemocytometer at time intervals. Data are expressed as mean S.D. from triplicate wells of one representative experiment, and signicant differences from control are shown by (* P < 0.05) and (** P < 0.01). Similar results were obtained in two independent experiments. Representative photographs of control cells (C) and cells treated with 4 mM vanillin (D) after 72 h exposure to vehicle or vanillin are also shown, original magnication 50.

Since the degree of inhibition of migration observed appears to be less than the inhibition of invasion obtained at the same concentrations of vanillin, it is possible that other effects may be involved in the inhibition of invasion. MMPs are a group of enzymes responsible for proteolysis of BM and ECM proteins in body and digest Matrigel in the invasion assay. Several anti-metastatic compounds inhibit MMPs with different mechanisms (Hidalgo and Eckhardt, 2001) including (1) direct inhibition of the enzymatic activity of MMPs, (2) interference in the activation of pro-MMPs and (3) reduced expression of MMP genes. The results from gelatin zymography and MMP inhibition assay suggest that vanillin has a direct inhibitory effect on the enzymatic activity of MMP-9, with little or no inhibitory effect on secretion of the enzyme. Since the vanillin structure is small and may not well t into the active site of the MMP-9 enzyme, its inhibitory effect on MMP-9 enzymatic activity may be due to binding to other sites on the enzyme molecule. Although most MMP inhibitors inhibit the enzymatic activity of MMPs by binding to the active site, a recent report showed that a avonoid, luteolin, inhibited enzymatic activity of MMP-9 by interaction with a site outside the active site, the so-called exosite (Ende and Gebhardt, 2004). Aspirin also showed a direct inhibitory effect on enzymatic activity of MMP-9 in lung adenocarcinoma with an unclear mechanism (Karna and Palka, 2002). So the inhibition of cancer invasion by vanillin should be a combination of an inhibitory effect on cell migration and an inhibitory effect on MMP-9 enzymatic activity.

Our in vitro data showed that vanillin decreased the growth rate of 4T1 cells without lethal damage to cell morphology. However, the concentration required for inhibiting growth was higher than that for inhibiting invasion. Since vanillin is rapidly metabolized to an inactive product, this may explain the decreased inhibitory effect of vanillin on primary tumor growth in vivo. Our data suggests that the aldehyde group of vanillin may be important for its anti-metastatic activity and the effect on cell growth, because the activities were absent in vanillic acid, which contains a carboxylic acid group instead of an aldehyde group. This provides important information for further study of the structure-activity relationship of vanillin-like compounds in the search for more potent compounds and mechanism of action. Anti-tumor activity of some vanillin-like small molecules, containing only one benzene ring with different substituent groups have been reported in breast cancer models. Perillyl alcohol inhibited breast cell migration (Wagner et al., 2002) and showed growth inhibitory effect on breast cancer cells in vitro and in vivo (Yuri et al., 2004). Salicylate inhibited cell growth and induced apoptosis in breast cancer cells in vitro (Sotiriou et al., 1999). Phenyl acetate decreased invasiveness of breast cancer cells (Vasse et al., 2001), induced apoptosis and inhibited tumor growth of breast cancer cells in vitro and in vivo (Adam et al., 1995). Caffeic acid and 3,4-dihydroxyphynylacetatic acid showed antiproliferative effect on human breast cancer cells (Kampa et al., 2004).

64

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765 tumor growth and metastasis in a syngeneic model of breast cancer. Cancer Res. 62, 46784684. Hagan, E.C., Hansen, W.H., Fitzhugh, O.G., Jenner, P.M., Jones, W.I., Taylor, J.M., Long, E.L., Nelson, A.A., Brouwer, J.B., 1967. Food avourings and compounds of related structure. II. Subacute and chronic toxicity. Food Cosmet. Toxicol. 5, 141157. Hake, C.L., Rowe, V.K., 1963. Ethers. In: Patty, F.A. (Ed.), Industrial Hygiene and Toxicology, vol. 2, second ed. Interscience Publishers, New York, NY, pp. 16551718. Hidalgo, M., Eckhardt, S.G., 2001. Development of matrix metalloproteinase inhibitors in cancer therapy. J. Natl. Cancer Inst. 93, 178193. Jiang, M.-C., Liao, C.-F., Lee, P.-H., 2001. Aspirin inhibits matrix metalloproteinase-2 activity, increases E-cadherin production, and inhibits in vitro invasion of tumor cells. Biochem. Biophys. Res. Commun. 282, 671677. Kampa, M., Alexaki, V.-I., Notas, G., Nii, A.-P., Nistikaki, A., Hatzoglou, A., Bakogeorgou, E., Kouimtzoglou, E., Blekas, G., Boskou, D., Gravanis, A., Castanas, E., 2004. Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action. Breast Cancer Res. 6, R63 R74. Karna, E., Palka, J.A., 2002. Inhibitory effect of acetylsalicylic acid on metalloproteinase activity in human lung adenocarcinoma at different stages of differentiation. Eur. J. Pharmacol. 443, 16. Keshava, C., Keshava, N., Ong, T., Nath, J., 1998. Protective effect of vanillin on radiation-induced micronuclei and chromosomal aberrations in V79 cells. Mutat. Res. 397, 149159. Kirwin, C.J., Galvin, J.B., 1993. Ethers. In: Clayton, G.D., Clayton, F.E. (Eds.), Pattys Industrial Hygiene and Toxicology, vol. 2, fourth ed. John Wiley & Sons, New York, pp. 445525 (Part A). Kohn, E.C., Liotta, L.A., 1995. Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res. 55, 18561862. Kumar, S.S., Priyadarsini, K.I., Sainis, K.B., 2004. Inhibition of peroxynitrite-mediated reactions by vanillin. J. Agric. Food Chem. 52, 139145. Lee, S.J., Sakurai, H., Oshima, K., Kim, S.H., Saiki, I., 2003. Antimetastatic and anti-angiogenic activities of a new matrix metalloproteinase inhibitor, TN-6b. Eur. J. Cancer 39, 16321641. Lloyd Jr., F.P., Slivova, V., Valachovicova, T., Sliva, D., 2003. Aspirin inhibits highly invasive prostate cancer cells. Int. J. Oncol. 23, 12771283. Michigami, T., Hiraga, T., Williams, P.J., Niewolna, M., Nishimura, R., Mundy, G.R., Yoneda, T., 2002. The effect of the bisphosphonate ibandronate on breast cancer metastasis to visceral organs. Breast Cancer Res. Treat. 75, 249258. Nakamura, E.S., Koizumi, K., Yamaura, T., Saiki, I., 2003. Anti-tumor angiogenic effect of a matrix metalloproteinase inhibitor MMI270. Anticancer Res. 23, 411418. Opdyke, D.L.J., 1977. Fragrance raw materials monographs. Vanillin. Food Cosmet. Toxicol. 15, 633638. Price, J.T., Thompson, E.W., 2002. Mechanisms of tumour invasion and metastasis: emerging targets for therapy. Expert Opin. Ther. Targets 6, 217233. Robinson, S.P., Langan-Fahey, S.M., Johnson, D.A., Jordan, V.C., 1991. Metabolites, pharmacodynamics, and pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer patient. Drug Metab. Dispos. 19, 3643. Shaughnessy, D.T., Setzer, R.W., DeMarini, D.M., 2001. The antimutagenic effect of vanillin and cinnamaldehyde on spontaneous mutation in Salmonella TA104 is due to a reduction in mutations at GC but not AT sites. Mutat. Res. 480481, 5569. Sotiriou, C., Lacroix, M., Lagneaux, L., Berchem, G., Body, J.-J., 1999. The aspirin metabolite salicylate inhibits breast cancer cells growth and their synthesis of the osteolytic cytokines interleukins-6 and -11. Anticancer Res. 19, 29973006. Tsuda, H., Uehara, N., Iwahori, Y., Asamoto, M., Iigo, M., Nagao, M., Matsumoto, K., Ito, M., Hirono, I., 1994. Chemopreventive effects of

In conclusion, our results showed that vanillin possesses anti-metastatic activity against breast cancer cells both in vitro and in vivo. Cell migration and ECM digestion are necessary for cancer invasion, so inhibition of both processes by vanillin would lead to the inhibition of cancer invasion in vitro and may be responsible, at least in part, for suppression of metastasis in vivo. Vanillin also showed inhibition of cell growth in vitro but could not suppress primary tumor growth in our animal experiments, so it is likely to be more useful as an anti-metastatic agent. The concentrations at which vanillin shows anti-metastatic effects in vitro are rather high (at mM levels). It is not known what concentrations of vanillin are reached in vivo under the conditions used in the present experiments. This will require further study. However, the dose of vanillin administered was also high (100 mg/(kg day)), and it is possible that there is accumulation of vanillin at the sites of action. Most importantly, the anti-metastatic effect of vanillin was observed in vivo at a dose that appears to be tolerated by the mice. Thus, the present data suggests that vanillin is an interesting compound for anticancer drug development, due to its safety and potency for the cytostatic therapy of cancer.

Acknowledgments This work was supported by a Japanese-Thai Collaborative Scientic Research Fellowship (JSPS-NRCT), 2003. We would like to thank Dr. Montip Tiensuwan, Department of Mathematics, Faculty of Science, Mahidol University, for her assistance with the statistical analysis.

References
Abiru, S., Nakao, K., Ichikawa, T., Migita, K., Shigeno, M., Sakamoto, M., Ishikawa, H., Hamasaki, K., Nakata, K., Eguchi, K., 2002. Aspirin and NS-398 inhibit hepatocyte growth factor-induced invasiveness of human hepatoma cells. Hepatology 35, 11171124. Abraham, D.J., Mehanna, A.S., Wireko, F.C., Whitney, J., Thomas, R.P., Orringer, E.P., 1991. Vanillin, a potential agent for the treatment of sickle cell anemia. Blood 77, 13341341. Adam, L., Crepin, M., Savin, C., Israel, L., 1995. Sodium phenylacetate induces growth inhibition and Bcl-2 down-regulation and apoptosis in MCF-7 ras cells in vitro and in Nude mice. Cancer Res. 55, 51565160. Anand-Apte, B., Zetter, B., 1997. Signaling mechanisms in growth factors-stimulated cell motility. Stem Cells 15, 259267. Akagi, K., Hirose, M., Hoshiya, T., Mizoguchi, Y., Ito, N., Shirai, T., 1995. Modulating effects of ellagic acid, vanillin and quercetin in a rat medium term multi-organ carcinogenesis model. Cancer Lett. 94, 113121. Durant, S., Karran, P., 2003. Vanillins-a novel family of DNA-PK inhibitors. Nucleic Acids Res. 31, 55015512. Ende, C., Gebhardt, R., 2004. Inhibition of matrix metalloproteinase-2 and -9 activities by selected avonoids. Planta Med. 70, 10061008. Fenteany, G., Zhu, S., 2003. Small-molecule inhibitors of actin dynamics and cell motility. Curr. Top. Med. Chem. 3, 593616. Guo, Y., Mazar, A.P., Lebrun, J.-J., Rabbani, S.A., 2002. An antiangiogenic urokinase-derived peptide combined with tamoxifen decreases

K. Lirdprapamongkol et al. / European Journal of Pharmaceutical Sciences 25 (2005) 5765 -carotene, -tocopherol and ve naturally occurring antioxidants on initiation of hepatocarcinogenesis by 2-amino-3-methylimidazo[4,5f ]quinoline in the rat. Jpn. J. Cancer Res. 85, 12141219. Vasse, M., Thibout, D., Paysant, J., Legrand, E., Soria, C., Crepin, M., 2001. Decrease of breast cancer cell invasiveness by sodium phenylacetate (NaPa) is associated with an increased expression of adhesive molecules. Br. J. Cancer 84, 802807. Wagner, J.E., Huff, J.L., Rust, W.L., Kingsley, K., Plopper, G.E., 2002. Perillyl alcohol inhibits breast cell migration without affecting cell adhesion. J. Biomed. Biotechnol. 2, 136140.

65

Welch, D.R., Lobl, T.J., Seftor, E.A., Wack, P.J., Aeed, P.A., Yohem, K.H., Seftor, R.E.B., Hendrix, M.J.C., 1989. Use of the membrane invasion culture system (MICS) as a screen for anti-invasive agents. Int. J. Cancer 43, 449457. Woodhouse, E.C., Chuaqui, R.F., Liotta, L.A., 1997. General mechanisms of metastasis. Cancer 80, 15291537. Yuri, T., Danbara, N., Tsujita-Kyutoku, M., Kiyozuka, Y., Senzaki, H., Shikata, N., Kanzaki, H., Tsubura, A., 2004. Perillyl alcohol inhibits human breast cancer cell growth in vitro and in vivo. Breast Cancer Res. Treat. 84, 251260.