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ScienceDirect Journal of Nutritional Biochemistry xx (2015) xxx – xxx

Alpha-carotene inhibits metastasis in Lewis lung carcinoma in vitro, and suppresses lung metastasis and tumor growth in combination with taxol in tumor xenografted C57BL/6 mice Yi-Zhen Liu a , Chih-Min Yang a , Jen-Yin Chen b, c , Junn-Wang Liao d, Miao-Lin Hu a, e,⁎ a

Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan b Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan c Department of the Senior Citizen Service Management, Chia Nan University of Pharmacy and Science, Tainan, Taiwan d Graduate Institute of Veterinary Pathology, National Chung Hsing University, Taichung, Taiwan e Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan

Received 5 June 2014; received in revised form 7 November 2014; accepted 12 December 2014

Abstract This study aimed to investigate the anti-metastatic activity of α-carotene (AC) in Lewis lung carcinoma (LLC) and in combination with taxol in LLCxenografted C57BL/6 mice. Cell culture studies reveal that AC significantly inhibited invasion, migration and activities of matrix metalloproteinase (MMP)-2, -9 and urokinase plasminogen activator but increased protein expression of tissue inhibitor of MMP (TIMP)-1, -2 and plasminogen activator inhibitor (PAI)-1. These effects of AC are similar to those of β-carotene at the same concentration (2.5 μM). AC (2.5 μM) also significantly inhibited integrin β1-mediated phosphorylation of focal adhesion kinase (FAK) which then decreased the phosphorylation of MAPK family. Findings from the animal model reveal that AC treatment (5 mg/kg) alone significantly decreased lung metastasis without affecting primary tumor growth, whereas taxol treatment (6 mg/kg) alone exhibited significant inhibition on both actions, as compared to tumor control group. AC treatment alone significantly decreased protein expression of integrin β1 but increased protein expression of TIMP-1 and PAI-1 without affecting protein expression of TIMP-2 and phosphorylation of FAK in lung tissues, whereas taxol treatment alone significantly increased protein expression of TIMP-1, PAI-1 and TIMP-2 but decreased protein expression of integrin β1 and phosphorylation of FAK. The combined treatment produced stronger actions on lung metastasis and lung tissues protein expression of TIMP-1, TIMP-2 and PAI-1. Overall, we demonstrate that AC effectively inhibits LLC metastasis and suppresses lung metastasis in combination with taxol in LLC-bearing mice, suggesting that AC could be used as an anti-metastatic agent or as an adjuvant for anti-cancer drugs. © 2015 Elsevier Inc. All rights reserved. Keywords: α-Carotene; β-Carotene; Lewis lung carcinoma; Metastasis; Taxol

1. Introduction Lung cancer is one of the most common diagnosed malignancies, among to 228,190 new cases and 159,480 deaths during 2012 [1]. The well known risk factors for lung cancer are cigarette smoking, radon gas exposure, second hand smoke and air pollution [2]. Lung cancer can be classified as small cell or non-small cell [3], and the treatments include surgery, radiation therapy, chemotherapy and targeted drug

Abbreviations: AC, α-carotene; BC, β-carotene; ERK, extracellular signal regulated kinase; FAK, focal adhesion kinase; FBS, fetal bovine serum; JNK, c-Jun NH2-terminal kinase; LLC, Lewis lung carcinoma; MMP, matrix metalloproteinase; PAI-1, plasminogen activator inhibitor-1; THF, tetrahydrofuran; TIMP, tissue inhibitor of MMP. ⁎ Corresponding author at: Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 402 ROC. Tel./fax: +886 4 2281 2363. E-mail address: [email protected] (M.-L. Hu). http://dx.doi.org/10.1016/j.jnutbio.2014.12.012 0955-2863/© 2015 Elsevier Inc. All rights reserved.

therapy based on the type and stage of cancer [4]. The major causes of death in lung cancer patients are tumor burden, infection and metastasis [5]. Tumor metastasis is a multi-steps and complex process that the metastatic spread of cancer cells from a primary tumor to distant sites involved invasion, migration, colonization and angiogenesis [6]. Therefore, inhibition of metastasis could be regarded as one of the therapeutic strategies for cancer treatment. Epidemiological studies have indicated that elevated intake of fruits and vegetables rich in carotenoids are associated with lowered risk of several chronic diseases, including cancer [7,8]. Carotenoids are tetraterpenoid compounds containing 40 carbon atoms that are synthesized by plants and certain photosynthetic organisms [9]. Carotenoids consist of two classes, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons without oxygen) [10]. α-Carotene (AC), whose structure is similar to βcarotene (BC), is the second common form of carotene with a β-ring and an ε-ring at the opposite end, and therefore the converting capacity of AC to vitamin A is about half that of BC [10]. The dietary sources of AC are yellow-orange and dark-green vegetables, such as

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carrots, sweet potatoes, pumpkin, and broccoli [11]. In addition, serum concentration of AC is around 0.12 μM in humans [12]. Several studies have indicated that AC exhibited anti-cancer activity in preclinical and clinical researches. For example, AC was shown to decrease proliferation of human prostate cancer PC-3, DU145 and LNCaP cells [13] and of human neuroblastoma GOTO cells [14]. In addition, AC was found to inhibit metastasis of rat ascites hepatoma AH109A cells [15] and human hepatocinoma SK-Hep-1 cells [16]. In animal carcinogenic models, Murakoshi et al. [17] demonstrated that oral administration with AC inhibits spontaneous liver tumor development, lung tumor formation induced by 4-nitroquinoline-1-oxide and glycerol, and the formation of skin papillomas induced by 12-O-tetradecanoyl-phorbol-13-acetate and 7,12-dimethylbenz[α]anthracene. Epidemiological study has also indicated that higher serum levels of AC are related to lowered risks of cardiovascular disease and cancer among US adults [11]. Taxol, also known as paclitaxel, was isolated from the bark of the Pacific yew tree (Taxus brevifolia) and has been shown to have anticancer activity against several types of cancer, including lung cancer [18]. The anti-cancer mechanism of taxol involved destroying normal microtubular dynamics essential for cell division, promoting the polymerization of tubulin and producing dysfunctional microtubules [19]. A randomized phase III clinical trial has indicated that taxol exhibits single-agent activity in non-small cell lung cancer and promotes survival rates in lung cancer patients [20]. Our previous study has demonstrated that AC inhibits cancer metastasis in human hepatocarcinoma SK-Hep-1 cells [16]. However, it is still unclear whether AC has similar inhibitory effects in lung cancer cells. Herein, we investigated the anti-metastatic effects of AC in comparison with BC and explored the possible mechanisms of AC underlying such action using highly invasive Lewis lung carcinoma (LLC). We further used LLC xenografted C57BL/6 mice to determine the in vivo anti-metastatic actions of AC in combination with or without taxol.

2.4. Tumor xenografted model C57BL/6 male mice (4 weeks old; 20–25 g) were obtained from BioLASCO Taiwan Co., Ltd (Yilan, Taiwan). The study protocol was approved by the Animal Research Committee of National Chung Hsing University (IACUC approval No. 100-87). Mice were housed in cages with controlled temperature (25±2°C) and humidity (65±5%) with 12-h light/dark cycles. After accommodation for 1 week, approximately 2×106 LLC cells (0.1 ml/mouse) were injected (s.c.) into the right flank of mice. Nine days after implantation, mice were randomly divided into four groups (n=7–8 for each group) as follows: Group 1, tumor control (tumor cell implantation, n=8); Group 2, AC (tumor implantation and oral supplementation with 5 mg/kg AC, n=7); Group 3, taxol (tumor implantation and i.p. injection with 6 mg/kg taxol, n=7); Group 4, AC+taxol (tumor implantation and oral supplementation with 5 mg/kg AC and i.p. injection with 6 mg/kg taxol, n=8). AC and taxol were given after the growth of an established tumor (typically, N50 mm3 tumor volume at start of treatment) for additional 3 weeks. During the accommodation and experimental periods, mice were supplied a standard rodent diet (Lab 5001, Purina Mills) and water ad libitum. The standard diet contains 59.8% carbohydrate, 23.4% protein, 4.5% crude fat without AC, as indicated by the supplier. The body weights of mice were measured weekly and the tumor growth was determined twice a week by direct measurement of tumor length and width using caliper, and tumor areas were calculated as the formula: π/4×length×width [22]. At the end of experiment, mice were euthanized with CO2 asphyxiation, and the primary tumors were isolated and weighed. All lung lobes of each mouse were observed and counted by naked eyes for measurement of lung metastasis. 2.5. Invasion and migration assay Invasion and migration assays were determined according to the published methods by Repesh [23] with minor modification using transwell chambers with 6.5 mm polycarconate filters of 8 μm pore size. The major difference between invasion and migration assay is that each filter for the invasion assay was pre-coated with 100 μl of 1:20 (v:v) diluted matrigel in cold DMEM to form a thin continuous film on the upper chamber. After pre-incubation with AC (0.5–2.5 μM) and BC (2.5 μM), LLC cells (5×104/400 μl for invasion and 8×104/400 μl for migration) were suspended in serum-free DMEM medium and were replaced in the top of the chamber. The lower chamber was added DMEM medium containing 10% FBS. After incubation of an additional 24 h for invasion and 6 h for migration, the cells on the upper chamber were completely wiped away with cotton swabs. The cells on the lower chamber were fixed with methanol for 10 min, stained with Giemsa, and then counted under a microscope. The cells in five randomly selected fields were photographed and the counts were averaged for each replicate. 2.6. Zymography assay

2. Materials and methods 2.1. Materials The solvents, such as tetrahydrofuran (THF), butylated hydroxyl toluene (BHT) and methanol were purchased from Merck (Darnstadi, Germany). The materials for cell culture, such as Dulbecco’s modified eagle medium (DMEM), non-essential amino acid, penicillin, sodium pyruvate, trypsin and fetal bovine serum (FBS) were purchased from GIBCO/BRL (Rockville, MD, USA). Taxol, gelatin, casein, and plasminogen were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies against extracellular signal regulated kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK), p38 MAPK proteins, focal adhesion kinase (FAK, Tyr397), and phosphorylated from of FAK rabbit monoclonal antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-integrin β1, anti-tissue inhibitor of matrix metalloproteinase (TIMP)-1 and -2, plasminogen activator inhibitor (PAI)-1 and β-actin rabbit monoclonal antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were purchased from Epitomics (Burlingame, CA, USA).

2.2. Preparation of AC and BC AC and BC were purchased from Wako (Osaka, Japan) and Calbiochem (Darmstadt, Germany), respectively, and the purity AC and BC was N97%, as claimed by the supplier. AC and BC were dissolved in THF containing 0.0025% BHT to form 10 mM stock solution. The stock solution was diluted with FBS at indicated ratio [21]. THF/BHT-FBS-AC or -BC was added to the culture medium at a calculated final concentration of 0.5–2.5 μM for AC and 2.5 μM for BC. THF at 0.2% (v/v) and FBS at 1.8% (v/v) served as the solvent control, which did not significantly affect the assays described below.

The activities of matrix metalloproteinase (MMP)-9, MMP-2 and urokinase plasminogen activator (uPA) in culture medium were determined using zymography assay based on the methods as reported by Leber and Balkwill [24] with minor modifications. Briefly, LLC cells (5×104 cells/ml) were incubated with AC (0.5–2.5 μM) or BC (2.5 μM) for 24 h in DMEM medium containing 2% FBS, and then incubated for an additional 24 h in serum free medium. The serum free medium was collected and electrophoresed (80 V; 120 min) in a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) containing gelatin for MMP-9 and -2 assay and casein-plasminogen for uPA assay. After electrophoresis, the gel was washed for 30 min in a washing buffer containing 2.5% (v/v) Triton X-100 at room temperature, and then incubated in reaction buffer containing 40 mM Tris–HCl, 10 mM CaCl2 and 0.01% NaN3 for 24 h at 37 °C. The gel was stained with Coomassie brilliant blue R-250 for 30 min followed by de-stained in mixture of 10% acetic acid (v/v) and 50% methanol (v/v). The relative activities of MMP-9, -2 and uPA were quantified using Matrox Inspector 2.1 software. 2.7. Western blotting Protein expressions in cell lysates and in lung tissues were determined by western blotting. In cell culture experiments, LLC cells were scraped at indicated incubation time and cellular proteins were extracted with RIPA buffer containing protease inhibitors. In animal experiments, lung tissues (0.05 g) were homogenized in cold Tissue Protein Extraction Reagent containing 1 mM phenylmethyl sulfonyl fluoride (1:1000, a total of 1 mL). The mixtures were subjected to a centrifugation of 10,000×g for 30 min at 4°C and the supernatant was collected. An amount of protein (40 μg) from supernatant was resolved by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. After blocking with TBS buffer (20 mM Tris–HCl, 150 mM NaCl, pH 7.4) containing 5% nonfat milk, the membrane was incubated with primary antibody followed by horseradish peroxidase-conjugated antimouse IgG, and then visualized using ECL chemiluminescent detection kit (Amersham Co, Bucks, UK). The relative density of protein expression was quantified by densitometry (Matrox Inspector 2.1 software).

2.3. Cell culture

2.8. Statistical analysis

The mouse Lewis lung carcinoma (LLC, BCRC 60050) cell line was obtained from Food Industry Research and Development Institute (Hsin Chu, Taiwan). Cells were cultured in DMEM containing 10% (v:v) FBS, 0.37% (w/v) NaHCO3, penicillin (100 kU/L), streptomycin (100 kU/L) in a humidified incubator under 5% CO2 and 95% air at 37°C.

Values are expressed as means±S.D. and analyzed using one way ANOVA followed by Fisher’s protected least significant difference test for comparisons of group means, when the F value was significant (Pb.05). All statistical analyses were performed using SPSS for Windows, version 10 (SPSS, Inc.); Pb.05 is considered statistically significant.

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3. Results 3.1. Effects of AC and BC on invasion and migration in LLC AC (0.5–2.5 μM) significantly and concentration-dependently inhibited invasion of LLC during 48 h of incubation, with an inhibition of 38.2% (Pb.05) for 12 h, 45% (Pb.05) for 24 h and 32.4% (Pb.05) for 48 h at AC 2.5 μM (Fig. 1A). Such inhibition was also observed in migration of LLC by AC (0.5-2.5 μM) treatment, with an inhibition of 33.5% (Pb0.5) for 12 h, 42.2% (Pb.05) for 24 h and 30.9% (Pb.05) for 48 h at AC 2.5 μM (Fig. 1B). In addition, BC has similar inhibitory extent on invasion and migration at 12, 24 and 48 h of incubation, as compared to AC at the same concentration (2.5 μM) (Fig. 1A and B). Under these conditions, neither AC (0.5–2.5 μM) nor BC (2.5 μM) affected cell proliferation of LLC (data not shown). According to the time course effect, the strongest inhibitory effect occurred at 24 h of incubation; therefore, we chose the incubation time of 24 h or b24 h for the following experiments. 3.2. Effects of AC and BC on activities of MMP-9, -2 and uPA in LLC Incubation of LLC with AC (0.5–2.5 μM) for 24 h significantly decreased activities of MMP-9, -2 and uPA in concentration-dependent manner, with an inhibition of 38% (Pb.05) for MMP-9, 32% (Pb.05) for MMP-2 and 34% (Pb.05) for uPA at 2.5 μM AC (Fig. 2A). BC also significantly reduced activities of MMP-9 and MMP-2 in a similar extent, as compared to AC at the same concentration (2.5 μM) (Fig. 2A). However, the inhibitory effect of BC on uPA activity was significantly lower than those of AC at the same concentration (2.5 μM) (Fig. 2A). 3.3. Effects of AC and BC on protein expression of TIMP-1, TIMP-2 and PAI-1 in LLC Incubation of LLC with AC (1 and 2.5 μM) for 24 h significantly increased protein expression of TIMP-1 and TIMP-2 in a concentration-dependent manner, with a promotion 35% (Pb.05) and 32% (Pb.05), respectively, at 2.5 μM AC (Fig. 2B). In addition, AC (0.5–2.5 μM) significantly increased PAI-1 protein expression in LLC and the most effective concentration was at 2.5 μM AC, with a promotion of 53% (Pb.05) (Fig. 2B). BC also significantly increased protein expression of TIMP-1, TIMP-2 and PAI-1 in a similar extent, as compared to AC at the same concentration (2.5 μM) (Fig. 2B). 3.4. Time-course effects of AC on expression of integrin β1-mediated signaling molecules in LLC Cells were incubated with AC (2.5 μM) for 15–180 min to measure the time course effect on expression of integrin β1-mediated signaling molecules, including FAK and MAPK family. Results reveal that AC (2.5 μM) transiently but significantly decreased protein expression of integrin β1 at 45 and 60 min, with a reduction of 31% (Pb.05) at 60 min of incubation (Fig. 3). We also found that AC (2.5 μM) significantly decreased phosphorylation of FAK during 180 min of incubation, and the most effective reduction occurred at 30–60 min, with an inhibition of 47% (Pb.05) at 45 min of incubation (Fig. 3). In addition, AC (2.5 μM) transiently but significantly inhibited phosphorylation of ERK 1 and 2 at 30–180 min of incubation, and the strongest inhibition occurred at 60 min of incubation, with an inhibition of 38% (Pb0.05) for ERK1 and 31% (Pb.05) for ERK2 (Fig. 3). Similarly, AC (2.5 μM) transiently but significantly suppressed phosphorylation of p38 at 45 and 60 min of incubation, with an inhibition of 22% (Pb.05) at 45 min of incubation (Fig. 3). Moreover, AC (2.5 μM) transiently but significantly inhibited phosphorylation of JNK1 at 30–180 min and JNK2 at 45 and 60 min of incubation, and the most effective inhibition was at 60 min of incubation, with an

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inhibition of 31% (Pb.05) and 24% (Pb.05), respectively (Fig. 3). In contrast, AC (2.5 μM) did not affect protein expression of FAK and MAPK family (Fig. 3). 3.5. Effects of AC and taxol alone and the combined treatment on body weights, primary tumor growth, and lung metastasis in LLC-bearing mice AC alone, taxol alone and combined treatment did not decrease final and relative body weight during entire experimental period as compared to tumor control mice, suggesting that AC and taxol have no toxicological effects in tumor-bearing mice (Table 1). AC treatment alone did not affect primary tumor growth (Fig. 4A upper panel and B; Table 1), whereas taxol treatment alone significantly inhibited primary tumor growth, as evidenced by decreased tumor areas (Fig. 4A upper panel and B) and tumor weights (Table 1), as compared to tumor control group. The combined treatment also significantly inhibited tumor growth in comparison with tumor control group, but the effect was not significantly different from that of the AC or taxol treatments alone (Fig. 4A upper panel and B). Both AC alone and taxol alone significantly decreased the numbers of metastatic foci in lung tissues, with an inhibition of 17% (Pb.05) and 36% (Pb.05), respectively, as compared to tumor control group (Fig. 4A, lower panel). The combined treatment produced stronger inhibition (49%) on lung metastasis than AC and taxol treatments alone (Pb.05) (Fig. 4A lower panel). 3.6. Effects of AC alone, taxol alone and combined treatment on protein expression of integrin β1, TIMP-1, TIMP-2 and PAI-1 as well as phosphorylation of FAK in lung tissues of LLC-bearing mice AC and taxol treatments alone significantly decreased protein expression of integrin β1, with an inhibition of 22.7% (Pb.05) and 22.5% (Pb.05), respectively, as compared to tumor control (Fig. 5). Taxol treatment alone significantly reduced phosphorylation of FAK in lung tissues (15.4%, Pb.05), whereas AC treatment alone had no significant effect (Fig. 5). However, the combined treatment did not further inhibit protein expression of integrin β1 and phosphorylation of FAK, as compared to taxol treatment alone (Fig. 5). AC treatment alone significantly increased protein expression of TIMP-1 and PAI-1, with an increase of 25.1% (Pb.05) and 57.9% (Pb.05), respectively, but did not affect TIMP-2 protein expression in lung tissues, as compared to those of tumor control (Fig. 5). In addition, TIMP-1, TIMP-2 and PAI-1 protein expression were increased by taxol alone treatment, with a promotion of 50.3% (Pb.05), 23.9% (Pb.05) and 32.6% (Pb.05), respectively, in comparison with tumor control group (Fig. 5). The combined treatment further enhanced these effects, with an increase of 71.2% (Pb.05) for TIMP-1, 44.5% (Pb.05) for TIMP-2, and 122.9% (Pb.05) for PAI-1, as compared to AC alone and taxol treatments alone (Fig. 5). 4. Discussion Our previous study has demonstrated that AC can inhibit the metastasis of human hepatocarcinoma SK-Hep-1 cells [16]. However, it is still unclear whether AC possesses similar effects on lung cancer which is also the major leading cause of death in cancer patients worldwide. The cell culture findings from the present study demonstrated that AC exhibited anti-metastatic activity in mouse lung cancer cells. The well-established animal model of LLC-bearing C57BL/6 mice can be used to investigate the primary tumor growth and lung metastasis [25]. In the present study, we report for the first time that AC effectively inhibited lung metastasis in LLC-bearing mice and enhanced the efficacy of taxol on lung metastasis when combination used.

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Fig. 1. Effects of α-carotene and β-carotene on invasion and migration in LLC. Cells were incubated with α-carotene (0.5–2.5 μM) and β-carotene (2.5 μM) for 12, 24 and 48 h before transwell chamber assay. (A) Images of cell invasion; (B) images of cell migration. Values are expressed as mean±S.D. from four separate experiments, means not sharing an alphabetic letter differ statistically, Pb.05.

Several possible mechanisms may be involved in the antimetastatic action of AC in LLC. One is that the imbalance between MMPs and TIMPs was caused by AC treatment. MMPs are zincdependent endopeptidases that can degrade all known components of extracellular matrix (ECM) [26]. MMPs are also involved in the regulation of many physiological and pathological processes, such as

embryonic development, wound healing, angiogenesis, apoptosis, arthritis, metastasis and cardiovascular disease [27,28]. Among these MMPs, MMP-2 and MMP-9 (also called gelatinase or collagenase IV) preferentially degrade the basal membrane and are deeply involved in tumor metastasis [29]. Overexpression of MMP-2 and MMP-9 is known to be one of the characteristics in highly metastatic tumors

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Fig. 2. Effects of α-carotene and β-carotene on activities of MMP-9, MMP-2 and uPA as well as on protein expression of TIMP-1, TIMP-2 and PAI-1 in LLC cells. Cells were incubated with α-carotene (0.5–2.5 μM) and β-carotene (2.5 μM) for 24 h before zymography and western blotting assays. (A) Images of MMP-9, MMP-2 and uPA activities; (B) Images of TIMP-1, TIMP-2 and PAI-1 protein expression. Values are expressed as mean±S.D. from four separate experiments; means not sharing an alphabetic letter differ statistically, Pb.05.

[30,31]. MMPs activities are mediated at least by three ways: transcriptional medication, proteolytic activation of the zymogen, and active enzyme inhibition [29]. TIMPs, endogenous inhibitors of MMPs, can bind the active site of MMPs to form non-covalent 1:1 stoichiometric complexes that prevent the MMPs degradation [32]. Among the TIMPs family, TIMP-1 and TIMP-2 can form a complex with pro-MMP-9 and pro-MMP-2, respectively, leading to inhibition of the activity of MMPs [33]. Herein, our cell culture data indicated that inhibition of MMP-2 and MMP-9 activity and promotion of TIMP-1 and TIMP-2 protein expression are involved in the anti-metastatic effect of AC in LLC cells. Another mechanism that may be involved in the anti-metastatic action of AC in LLC is the inhibition of the uPA system. The major components in the uPA system, such as uPA and its endogenous inhibitor PAI-1, have been shown to play a critical role in cancer cell invasion and migration [34]. uPA can aid the conversion of inactive plasminogen to active plasmin leading to induced the formation of active MMPs [35]. Several studies have indicated that overexpression of uPA is correlated with the progression of tumor and poor prognosis in non-small lung cancer [36] and breast cancer [37]. Therefore, inhibition of uPA catalytic activity can reduce cancer metastasis. PAI-1, the endogenous inhibitor of uPA, can react with uPA forming a stable complex with a 1:1 stoichiometry [38]. The roles of PAI-1 in tumor metastasis are contradictory in the literature. On the one hand, PAI-1 can inhibit uPA activity leading to reduction of tumor metastasis [38]; on the other, PAI-1 can facilitate tumorigenesis and angiogenesis [39]. Our findings appear to support the former action because AC promoted PAI-1 expression and attenuated uPA activity which can result in reduction of active MMP-2 and MMP-9 formation and lead to inhibition of tumor metastasis. Integrins, a cluster of transmembrane receptors [40], are noncovalently heterodimers containing α and β subunits [41]. There are

18α and 8β subunits that can congregate into 24 different receptors with diverse binding characters in vertebrates [42]. Among them, integrin β1 is responsible for regulation of cell-cell and cell-ECM interactions followed by mediation of cytoskeleton formation leading to affecting cell proliferation and migration [43,44]. Overexpression or activation of integrin β1 was shown to promote tumor metastasis and to serve as a biomarker in metastatic cancer cells, including human lung cancer A549 cells [45] and human melanoma MDA-M435 cells [46]. FAK, a non-receptor tyrosine kinase, is found in the focal adhesions that adhering to ECM and plays a critical role in regulating signaling [47]. Integrin-generated signals can induce tyrosine phosphorylation of FAK which help transduction of signals to downstream MAPK cascade resulted in promotion of metastasis and cytoskeletal rearrangement in cancer cells [48,49]. In addition, attenuation of FAKmediated ERK1/2 phosphorylation leading to reduction of MMP-9 activity was shown to involve in inhibition of fibronectin-induced metastasis in human lung cancer A549 cells [50]. MAPK family consists of three members, including ERK1/2, p38 and JNK, and activation of MAPK requires phosphorylation of conserved tyrosine and threonine residues followed by translocation to the nucleus leading to activation of transcription factors [51]. In addition, MAPK has been shown to play an important role in metastasis through mediating cell migration and ECM degradation [51]. Our present findings indicate that AC significantly but transiently inhibited protein expression of integrin β1 and phosphorylation of FAK and MAPK family, suggesting that AC inhibits invasion and migration via attenuation of integrin β1mediated phosphorylation of FAK, followed by inhibition of ERK/ p38/JNK pathways leading to inhibition of MMP-2 and MMP-9 activity in LLC (Fig. 6). Several studies have indicated that AC possessed stronger potency in inhibiting proliferation in human prostate cancer cells [13] and human neuroblastoma GOTO cells than those of BC at the same

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Fig. 3. Time course effects of α-carotene on protein expression of integrin β1 as well as on the phosphorylation and protein expression on FAK and MAPK family (ERK1/2, p38 and JNK 1/2) in LLC. Cells were treated with α-carotene (2.5 μM) for 15–180 min before western blot assay. Values are expressed as mean±S.D. from four separate experiments; means not sharing an alphabetic letter differ statistically, Pb.05.

concentration [14]. In animal models, AC has more effective than BC against tumorigenesis in liver, lung, skin and colon, when AC and BC were orally supplied to mice or rats at the same doses [17,52]. Our previous study has also shown that the anti-metastatic effects of AC

Table 1 Effects of α-carotene alone, taxol alone and combined treatment on final body weights, relative body weights and tumor weights in LLC-bearing C57BL/6 mice 1. Groups

Tumor control α-Carotene Taxol Combined treatment

n

8 7 7 8

Body weights 2 Initial

Final

25.0±1.8 24.9±1.1 25.1±0.9 24.7±1.4

28.0±1.0a 30.2±0.8b 29.8±2.7b 27.9±0.8a

Relative body weights 2

Tumor weights

22.6±0.8a 24.1±0.9b,c 25.0±1.3c 22.9±0.6a,b

6.3±1.6a 6.3±0.8a 4.9±2.2b 4.9±1.0b

1 LLC cells (2×106 cells/100 μl) were injected (s.c.) once to C57BL/6 mice for 9 days, and mice were administrated orally with α-carotene (5 mg/kg), taxol (6 mg/kg) or their combination via intraperitoneal injection twice a week for an additional 21 days. Values are mean±S.D., n=7–8; means in a column not sharing a superscript alphabetic letter differ (Pb.05). 2 Body weights were measured once every week, and the relative body weights were obtained by subtracting tumor weights from final body weights at the end of the experiment.

were more effective than those of BC at the same concentration in human hepatocarcinoma SK-Hep-1 cells [16]. In addition, Schwartz and Shklar [53] have found that BC, but not 13-cis retinoic acid, inhibited tumor progression of oral carcinoma. These findings suggested that the anti-cancer action of AC and BC is not associated with their provitamin A activities. Our cell culture findings support this notion that AC, which has lower conversion rate to vitamin A than BC [10], and BC have similar inhibition on cancer metastasis at the same concentration (2.5 μM) in LLC. In many cell culture and animal studies, the growth and metastasis of cancer cells can be suppressed by many phytochemicals, but they are rarely applied for clinical cancer treatment [54]. Several phytochemicals, such as curcumin [55], genistein [56] and quercetin [57], have been shown to enhance the anti-cancer efficacy of available anticancer drugs when combined used. Taxol, a well-known anti-cancer drug, has been used for inhibition of tumor growth and promotion of survival rates in non-small cell lung cancer patients [20]. The dose of Taxol used in our animal study was based on that of a previous report that had employed the same tumor xenographted mouse model [58] in which taxol (paclitaxol) injected i.p. at 6 mg/kg body weight in C57BL/6 mice implanted with LLC cells was found to inhibit tumor volume by 26.2% (Pb.05) after a 15-day treatment. In the present

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Fig. 4. Effects of α-carotene, taxol and combined treatment on primary tumor growth and lung metastasis in LLC xenografted C57BL/6 mice. LLC cells (2×106 cells/100 μl) were injected (s.c.) once to C57BL/6 mice for 9 days, and mice were administrated orally α-carotene (5 mg/kg) and injected i.p. taxol (6 mg/kg) via intraperitoneally injection or combined treatment twice a week for an additional 21 days. (A) Macroscopic observation of primary tumor tissues (upper panel) and lung tissues (lower panel). (B) Effects of α-carotene, taxol and combined treatment on primary tumor areas and the formula for calculating the tumor areas is π/4×length×width. Values are mean±S.D., n=7–8; means not sharing an alphabetic letter differ, Pb.05.

study, we found that AC in combination with taxol significantly enhanced the inhibition of lung metastasis and promoted TIMP-1, TIMP-2 and PAI-1 protein expression in lung tissues, as compared to AC alone and taxol treatments alone. One question to ask about the present study is whether the concentrations of AC and BC used in cell cultured study as well as the dose of AC used in animal model have any relevance in humans. The physiological plasma concentrations of AC and BC are around 0.1 μM and 0.42 μM, respectively, in health populations. Micozzi et al. [59] have reported that plasma levels increased from 0.08 to 0.97 μM for AC and from 0.3 to 7.9 μM for BC in health men after consuming carrots (272 g/d) or pure BC capsule (30 mg/d), respectively, for 6 weeks. Therefore, the concentration of AC (0.5–2.5 μM) and BC (2.5 μM) used in cell culture experiments are supra-physiological and physiological level, respectively. In our animal model, the mice were orally administrated with 5 mg/kg AC three times per week for 21 days. The AC dose (5 mg/kg) can be translated into 2.14 mg/kg per day (i.e. 5 mg×3÷7). A formula is available for converting animal dose to human equivalent dose (HED) in mg/kg, i.e., multiply the animal dose

in mg/kg per day by 0.081 [60]. An HED of 10.4 mg/60-kg person per day was obtained by the equation: 2.14 mg/kg per day×0.081×60 kg. The fresh carrots contains 35.0 μg AC/g [61]; therefore, the calculated HED for AC is achievable for human supplementation, i.e., intake of 300 g carrots per day. In conclusion, the cell culture findings have indicated that the anti-metastatic action of AC involves the mediation of gene expression (i.e., uPA system and balance of MMPs and TIMPs) and signaling pathway (i.e., integrin β1-FAK-MAPK cascade) related to invasion and migration. The results from animal study have demonstrated AC inhibits lung metastasis and enhances the inhibitory effect of taxol on lung metastasis in LLC xenografted C57BL/6 mice. Further studies are needed to determine the potential of AC as an anti-metastatic agent or an adjuvant for anti-cancer drugs.

Conflict of interest statement We declare no conflict of interest involved in this study.

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References

Fig. 5. Effects of α-carotene, taxol, and the combined treatment on protein expression of integrin β1, TIMP-1, TIMP-2 and PAI-1 as well as phosphorylation of FAK. LLC cells (2×106 cells/100 μl) were injected (s.c.) once to C57BL/6 mice for 9 days, and mice were administrated orally with α-carotene (5 mg/kg) and injected i.p. with taxol (6 mg/kg) or the combined treatment twice a week for an additional 21 days. Values are mean± S.D., n=3–4; means not sharing an alphabetic letter differ, Pb.05.

Acknowledgement This research was supported in part by the Ministry of Education, Taiwan, ROC, under the ATU plan and NSC101-2320-B-039-007-MY3 from the National Science Council, Executive Yuan, Taiwan.

Fig. 6. A proposed schematic diagram for the mechanistic action of α-carotene on LLC metastasis in vitro.

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