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Antimetastatic activity of Sulforaphane
Life Sciences 78 (2006) 3043 – 3050 www.elsevier.com/locate/lifescie
Antimetastatic activity of Sulforaphane P. Thejass, Girija Kuttan ⁎ Department of Immunology, Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala State 680555, India Received 3 September 2005; accepted 2 December 2005
Abstract The effect of Sulforaphane on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice by three different modalities of administration—simultaneous, prophylactic and after tumour developed. Of this simultaneous mode of Sulforaphane administration was found to be most effective. There was 95.5% inhibition of lung tumour nodule formation and 94.06% increase in the life span of metastatic tumour bearing animals. Highly elevated levels of lung hydroxyproline, lung uronic acid, lung hexosamine, serum sialic acid and serum γ-glutamyl transpeptidase (GGT) in the metastatic control animals was found to be significantly lowered in the Sulforaphane treated animals. Histopathological analysis of lung tissues also correlated with these results. In the in vitro system Sulforaphane showed a significant inhibition in the invasion of B16F-10 melanoma cells across the collagen matrix. 3H-thymidine proliferation assay showed that Sulforaphane could inhibit the proliferation of B16F-10 melanoma cells in vitro. Gelatin zymographic analysis showed that Sulforaphane could inhibit the activation of matrix metalloproteinases. These findings suggest that Sulforaphane reduced the invasion of B16F-10 melanoma cells by the inhibition of activation of matrix metalloproteinases, thereby inhibiting lung metastasis. © 2006 Published by Elsevier Inc. Keywords: B16F-10 melanoma cells; Invasion; Matrix metalloproteinases; Metastasis; Proliferation; Sulforaphane
Introduction Metastasis or secondary neoplastic growth defines malignancy and is a major contributor to cancer mortality. The prognosis of cancer is mainly determined by the invasiveness of the tumour and its ability to metastasize. The metastatic process consists of several sequential events including escape of cancer cells from the original tumour, intravasation and dissemination through blood and lymphatic vessels, arrest in the microvasculature of the target organs, extravasation and proliferation at a new site (Fidler, 1978; Nicolson, 1982; Liotta, 1984; Netland and Zetter, 1989). Since basement membrane and extracellular matrix provide the main physical barriers to cancer cell invasion; proteolytic degradation of these structures has been proposed to be important in the metastatic process (Liotta, 1986a,b; Liotta et al., 1980). Any drug, which can inhibit one of the steps in the cascade, will be useful in the inhibition of tumour metastasis. Although there are several drugs available to control cancer growth in humans, there are no drugs presently available ⁎ Corresponding author. Tel.: +91 487 2307950; fax: +91 487 2307868. E-mail address: [email protected] (G. Kuttan). 0024-3205/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.lfs.2005.12.038
to specifically inhibit the metastasis of cancer cells. This is due to the fact that the cancer cells in different metastases and even in single metastases may respond differently to radiotherapy or chemotherapy. Isothiocyanates, the enzymatic hydrolysis product of glucosinolates, are naturally occurring constituents of cruciferous vegetables. Many isothiocyanates protect against chemically induced tumours in a variety of animal organs (Zhang and Talalay, 1994; Hecht, 1995). Phenyl and benzyl isothiocyanates inhibited carcinogenesis induced by carcinogens such as diethyl nitrosamine, dimethyl benzo (a) anthracene or benzo (a) pyrene (Wattenberg, 1987). The antimetastatic activity of Allyl isothiocyanate (AITC), and Phenyl isothiocyanate (PITC) has already been reported from our laboratory (Manesh and Kuttan, 2003). Sulforaphane, an isothiocyanate rich in broccoli (Brassica oleracea), is an inducer of phase II enzymes, which enhances detoxification of carcinogens, a mechanism contributing to anticarcinogenic activity (Zhang et al., 1992). Experiments in animal models showed that Sulforaphane protected the mouse forestomach against the neoplastic effects of B (a) P (Fahey et al., 2002). In the present study, the effect of Sulforaphane on the
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inhibition of pulmonary metastasis induced by B16F-10 melanoma cells in C57BL/6 mice was evaluated with special emphasis on the mechanism of action using in vitro models. Materials and methods Animals
histopathological analysis. The rest of the six animals in each group were observed for their survival. The mortality of the animals was observed and the percentage increase in life span (%ILS) was calculated using the formula %ILS = T − C / C × 100, where T represents the number of survival days of treated animals and C represents the number of survival days of control animals.
C57BL/6 mice (20–25 g body wt, 6–8 weeks old males) were purchased from National Institute of Nutrition, Hyderabad, India. The animals were fed with mouse chow (Sai Feeds, India) and water ad libitum. All animal experiments were conducted according to the rules and regulations of the Animal Ethics Committee, Govt. of India.
Histopathological analysis Lung tissues were fixed in 10% formalin, dehydrated in different concentrations of alcohol and embedded in paraffin wax. Sections (4 μm) were stained with eosin and hematoxylin.
Cell line
Determination of the cytotoxic activity of Sulforaphane towards B16F-10 melanoma cells in culture by MTT assay B16F-10 melanoma cells were seeded (5000 cells/well) in 96-well flat bottomed titre plate and incubated for 24 h at 37 °C in 5% CO2 atmosphere. Different concentrations of Sulforaphane (1–50 μg/ml) were added and incubated further for 48 h. Before 4 h of completion of incubation, 20 μl MTT (5 mg/ml) was added (Cole, 1986; Campling et al., 1991). Percentage of dead cells was determined using an ELISA plate reader set to record absorbance at 570 nm.
B16F-10 melanoma, a highly metastatic cell line was obtained from the National Centre for Cell Sciences, Pune, India. The cells were maintained in DMEM, supplemented with 10% FCS and antibiotics. Chemicals Sulforaphane and hydroxyproline were purchased from Sigma Chemicals, St.Louis, MO, USA. N-acetyl neuraminic acid was purchased from Sisco Research Laboratory, Mumbai. All other reagents were of analytical grade. Drug administration Sulforaphane was suspended in phosphate buffered saline (PBS, pH-7.4) and used for all the experiments. Determination of the antimetastatic activity in the in vivo system Determination of the effect of Sulforaphane on lung tumour nodule formation and rate of survival C57BL/6 mice were divided into 4 groups (14 nos/group). All the animals were injected with B16F-10 melanoma cells (106 cells/ animal) through lateral tail vein. (Liotta, 1986a,b). To three groups of animals Sulforaphane was administered intraperitoneally at a dose of 500 μg/kg body wt for 10 consecutive days in three different modalities—simultaneously with metastatic tumour cells (Group I); 10 days prior to metastatic tumour induction (Group II) and 10 days after tumour induction (Group III). Group IV animals were kept as untreated metastatic tumour bearing control. Eight animals from each group were sacrificed on the 21st day after tumour challenge, lungs were excised and blood was collected. Lungs were used for morphological examinations of metastatic tumour nodules and for the estimation of collagen hydroxyproline (Bergman and Loxley, 1940), hexosamine (Elson and Morgan, 1933) and uronic acid (Bitter and Muir, 1962) contents. Serum was separated from the blood and used for determining the sialic acid (Skoza and Mohos, 1976) and γ-glutamyl transpeptidase (GGT) (Szasz, 1976) levels. A portion of the lung was used for
Determination of antimetastatic activity using in vitro models
Collagen matrix invasion assay The invasion assay was carried out in modified Boyden chambers as described by Albini et al (Albini et al., 1987). The lower compartment of the chamber was filled with serum free DMEM and a polycarbonate filter coated with 25 μg Type I collagen was placed above this. B16F-10 melanoma cells (105 cells/150 μl DMEM) were then seeded on to the upper chamber in the presence and absence of Sulforaphane (1, 2 and 5 μg/ml) and incubated at 37 °C in 5% CO2 atmosphere for 10 h. After incubation, the filters were removed, fixed with methanol and stained with crystal violet. Cells migrating to the lower surface of the polycarbonate filters were counted under a microscope. The results were expressed as percentage inhibition of invasion. Determination of the effect of Sulforaphane on the inhibition of tumour cell proliferation (3H-thymidine incorporation assay) B16F-10 melanoma cells (5000 cells/well) were plated in a 96-well culture plate and incubated at 37 °C in 5% CO2 atmosphere. After 24 h, various concentrations of Sulforaphane (1, 2 and 5 μg/ml) were added and further incubated for 48 h. 3 H-thymidine was added to each well (1 μCi /well) and incubation was continued for additional 18 h. After completing incubation, the plates were centrifuged and the culture supernatant was removed, the cells were washed three times with PBS and then treated with ice cold PCA for 15 min. The resulting precipitate was dissolved in 0.5 N NaOH and was added to the scintillation fluid and kept overnight in the dark.
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The radioactivity was counted using a Rack Beta liquid scintillation counter.
Table 2 Effect of Sulforaphane on the lung biochemical parameters of metastases bearing animals
Gelatin zymography
Treatment
Hydroxyproline (μg/mg protein)
Uronic acid (μg/100 mg tissue wet wt)
Hexosamine (mg/100 mg tissue dry wt)
Normal Control Sulforaphane (500 μg/kg body wt) Prophylactic Simultaneous Developed
1.19 ± 0.10 21.91 ± 1.16
32.20 ± 2.00 352.62 ± 8.33
0.40 ± 0.10 4.12 ± 0.29
8.95 ± 0.66⁎ 4.99 ± 0.35⁎ 14.66 ± 0.70⁎
112.93 ± 5.10⁎ 85.14 ± 6.09⁎ 162.81 ± 7.13⁎
1.39 ± 0.17⁎ 0.90 ± 0.12⁎ 2.09 ± 0.15⁎
SDS-PAGE was performed with 5% gelatin incorporated in the separating gel (Billings et al., 1991). B16F-10 melanoma cells of subconfluent cultures were incubated with serum free medium for 24 h at 37 °C in 5% CO2 atmosphere. The conditioned medium was then collected and subjected to zymographic analysis. Fifty microlitres of sample (equivalent to 100 μg protein) was activated with 5 μl trypsin solution (75 μg/ml) in the presence and absence of Sulforaphane (2 and 5μg/ml) in 0.1 M Tris–HCl, 10 mM CaCl2 buffer (pH-8.0) and incubated for 1 h at room temperature. Samples were mixed with an equal volume of 2 × sample buffer and loaded on to 11% polyacrylamide gels containing 5% gelatin. Electrophoresis was carried out at 4 °C with constant current of 2 mA/tube until the tracking dye reached the periphery. The gels were then washed with 2% Triton X-100 in 0.1 M Tris–HCl, 10 mM CaCl2 at 37 °C for 18 h followed by staining with Gelcode Blue stain reagent for 2 h. Gels were destained to visualize the clear area against the dark background. Statistical analysis Values were expressed as mean ± S.D. The statistical analysis was done by using one-way ANOVA followed by Dunnett's test. Results Determination of antimetastatic activity in the in vivo system
The lungs were dissected out and assayed different biochemical parameters on the 22nd day after induction of B16F-10 melanoma cells (106 cells) through the lateral tail vein. For Prophylactic group of animals, drug administration started 10 days prior to tumour challenge, simultaneous group received the drug simultaneously with tumour challenge and developed group were given drug, 10 days after tumour challenge. Values are mean ± S.D., ⁎p b 0.001.
1994). The three different modalities of compound administration were found to be significantly effective. Of this simultaneous mode of administration produced maximum inhibition of 95.5% followed by prophylactic mode of administration (90.5%) and administration after tumour development (82.5%). Administration of Sulforaphane significantly increased the life span of tumour bearing animals (Table 1). The life span was highly enhanced in the case of simultaneous mode of administration (94.06%). Prophylactic administration enhanced the life span by 62.17% where as administration after tumour development increased the lifespan by 37.85%. Effect of Sulforaphane on the biochemical parameters of the metastasis bearing animals
Effect of Sulforaphane on the lung tumour nodule formation Metastatic tumour bearing animals treated with Sulforaphane showed significant reduction in tumour nodule formation. (Table 1). Metastatic control animals had massive tumour growth and were assigned an arbitrary number of 250 (Hill et al., Table 1 Effect of Sulforaphane on lung colonization of B16F-10 melanoma cells and survival of animals Treatment
Number of lung tumour nodules
% inhibition of nodule formation
%ILS
Control Sulforaphane (500 μg/kg body wt) Prophylactic Simultaneous Developed
250 a
–
–
23.71 ± 4.11 ⁎ 11.25 ± 2.21 ⁎ 43.87 ± 6.85 ⁎
90.51 95.50 82.45
77.80 94.06 62.17
The lungs were dissected out and observed for metastases on the 22nd day after induction of B16F-10 melanoma cells (106 cells) through the lateral tail vein. Prophylactic group of animals received Sulforaphane prior to tumour induction (10 doses, i.p.), simultaneous group received the drug simultaneously with tumour induction where as developed group received drug after 10 days of tumour induction. Values are mean ± S.D. a An arbitrary number of 250 is given for massive number of tumour nodules. ⁎ p b 0.001.
Lung collagen hydroxyproline Effect of Sulforaphane on the lung biochemical parameters is presented in Table 2. Control metastatic tumour bearing animals showed an increased level of lung collagen hydroxyproline (21.91 ± 1.16 μg/mg protein), which was significantly reduced in animals treated with Sulforaphane by the simultaneous mode Table 3 Effect of Sulforaphane on serum sialic acid and GGT levels of B16F-10 melanoma bearing animals Treatment
Sialic acid (μg/mL serum)
GGT (nmol p-nitroaniline/mL serum)
Normal Control Sulforaphane (500 μg/kg body wt) Prophylactic Simultaneous Developed
21.30 ± 1.50 109.68 ± 1.67
24.00 ± 0.17 108.26 ± 2.16
59.51 ± 1.29 ⁎ 35.13 ± 0.90 ⁎ 92.88 ± 1.23 ⁎
54.93 ± 1.58 ⁎ 39.15 ± 1.13 ⁎ 76.58 ± 0.90 ⁎
The serum was collected on 22nd day of tumour challenge by B16F-10 melanoma cells through lateral tail vein and assayed for serum biochemical parameters. Values are mean ± S.D. ⁎ p b 0.001.
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of administration (4.99 ± 0.35 μg/mg protein). Prophylactic mode of administration was also found to be effective with a reduced level of lung collagen hydroxyproline content (8.95 ± 0.66 μg/mg protein). Treatment of Sulforaphane after tumour development also produced considerable reduction of lung collagen hydroxyproline (14.66 ± 0.7 μg/mg protein), but not as much as the other two modalities. Lung hexosamine content Lung hexosamine level was highly increased in control metastatic tumour bearing animals (4.12 ± 0.29 mg/100 mg tissue dry wt) compared to the normal animals (0.4 ± 0.1 mg/100 mg tissue dry wt). When Sulforaphane was administered simultaneously with the tumour cells this elevated level was reduced to 0.9 ±
0.12 mg/100 mg tissue dry wt and when it was administered prophylactically the level was 1.39 ± 0.17 mg/100 mg tissue dry wt. Administration of Sulforaphane after tumour development also reduced this level to 2.09 ± 0.15 mg/100 mg tissue dry wt (Table 2). Uronic acid levels of the lung In control metastatic tumour bearing animals, the lung uronic acid level was drastically elevated (352.62 ± 8.33 μg/100 mg tissue wet wt), as compared to normal level (32.2 ± 2 μg/100 mg tissue wet wt) which was significantly reduced after the simultaneous administration of Sulforaphane (85.14 ± 6.09 μg/100 mg tissue wet wt). Considerable reduction in the lung uronic acid level was also obtained after prophylactic (112.93 ± 5.1 μg/
Fig. 1. Histopathological analysis of lungs from metastatic tumour bearing mice. (a) Normal; (b) control; (c) treated with Sulforaphane by simultaneous treatment modality; (d) treated with Sulforaphane by prophylactic treatment modality; (e) treated with Sulforaphane after tumour development.
P. Thejass, G. Kuttan / Life Sciences 78 (2006) 3043–3050 Table 4 Cytotoxicity of Sulforaphane towards B16F-10 melanoma cells in culture Concentration (μg/ml) of Sulforaphane
Percentage of cytotoxicity
1 2 5 10 20 25 50
0.4 2.4 3.6 6.4 17.2 32.4 42
B16F-10 melanoma cells were incubated with different concentrations (1–50 μg/ml) of Sulforaphane. Percentage of cytotoxicity was determined using MTT assay.
100 mg tissue wet wt) and developed (162.81 ± 7.13 μg/100 mg tissue wet wt) modalities of administration when compared to control group (Table 2). Serum sialic acid level The effect of Sulforaphane on the serum biochemical parameters is presented in Table 3. The serum sialic acid level of control metastatic tumour bearing animals was highly increased (108.26 ± 1.92 μg/ml serum) as compared to normal (21.3 ± 1.5 μg/ml serum). Here also the simultaneous adminis-
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tration of Sulforaphane significantly reduced the elevated serum sialic acid level to 35.13 ± 0.9 μg/ml serum, followed by prophylactic modality (59.51 ± 1.2 μg/ml serum) and in developed modality it was reduced only to 92.88 ± 1.23 μg/ml serum. Serum γ-glutamyl transpeptidase (GGT) level Here again, the highly elevated level of GGT in the serum of control metastatic tumour bearing animals (108.26 ± 1.92 n mol P-nitroaniline/ml serum) as compared to normal animals (24 ± 0.17 n mol P-nitroaniline/ml serum) was significantly reduced by the simultaneous mode of administration of Sulforaphane (39.15 ± 1.13 n mol P-nitroaniline/ml serum), followed by prophylactic mode of administration (54.93 ± 1.58 n mol Pnitroaniline/ml serum) where as in animals treated after tumour development this level was 76.58 ± 0.9 n mol P-nitroaniline/ml serum (Table 3). Histopathological analysis of lungs The hematoxylin and eosin stained sections of lung tissues are shown in Fig. 1(× 100). The lungs of control metastatic tumour bearing animals (Fig. 1b) showed prominent tumour
Fig. 2. The effect of Sulforaphane on the collagen matrix invasion. (a) Untreated control; (b) treatment with Sulforaphane (1 μg/ml); (c) treatment with Sulforaphane (2 μg/ml); (d) treatment with Sulforaphane (5 μg/ml).
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nodules around terminal bronchiole. These tumour nodules are characterized by polygonal tumour cells with prominent nucleolus, intracellular melanin deposition and clear areas of necrosis. This massive infiltration of the neoplastic cells around the main bronchioles, which make alveolar passages indistinguishable, extended to the pleura. This together with fibrosis reduces alveolar space, which in turn leads to reduced vital capacity. Simultaneous administration of Sulforaphane showed significant reduction in tumour mass. Alveoli and pleura were tumour free, alveolar passage lined with healthy ciliated columnar epithelial cells (Fig. 1c) and almost similar to normal lung (Fig. 1a). Considerable reduction of tumour mass was also observed in both prophylactic and developed modalities of administration (Fig. 1d and e). Determination of antimetastatic activity in the in vitro system Cytotoxic activity of Sulforaphane towards B16F-10 melanoma cells by MTT assay Cytotoxicity of Sulforaphane towards B16F-10 melanoma cells in culture is shown in Table 4. At 1–5 μg/ml Sulforaphane was found to be non-toxic to B16F-10 melanoma cells and these concentrations were used for further in vitro experiments. Collagen matrix invasion assay Metastatic B16F-10 melanoma cells show highly invasive property through the collagen matrix. Very high numbers of cells were found in the lower surface of the polycarbonate membrane, but administration of Sulforaphane produced significant inhibition in the invasion of the collagen matrix by the tumour cells in a dose dependent manner. At a concentration of 5 μg/ml Sulforaphane significantly inhibited the invasion of B16F-10 melanoma cells by 92.72% where as at 2 μg/ml and 1 μg/ml the percent of inhibition of invasion was found to be 61.18% and 36.42%, respectively (Fig. 2). Effect of Sulforaphane on tumour cell proliferation Proliferation rate was determined by the 3H-thymidine incorporation by the DNA of B16F-10 melanoma cells. Thymidine incorporation is proportional to the potential of the cells to synthesize DNA. Proliferation was expressed as radioactive count per minute. Untreated B16F-10 cells had very high rate of proliferation (4842.33 ± 230.7 cpm). Administration of SulforTable 5 Effect of Sulforaphane on tumour cell proliferation Treatment
Radioactive count/minute (CPM)
% inhibition
Untreated control Sulforaphane 1 μg 2 μg 5 μg
4842.33 ± 230.70
–
3316.66 ± 117.55 2840.66 ± 27.57 1662.00 ± 117.72
31.50 41.33 65.66
B16F-10 melanoma cells (5 × 103 cells/well) were grown in 96 well flat bottom plate After 24 h various concentrations of Sulforaphane were added and incubation was continued for 48 h. After incubation, 3H-thymidine was added to each well (1 μ Ci /well) and incubation was continued for 18 h. Cells were lysed and radioactivity was counted by using Rack Beta liquid scintillation counter. Values are mean ± S.D.
Fig. 3. Effect of Sulforaphane on the activation of matrix metalloproteinases. (A) Conditioned medium without trypsin activation; (B) trypsin activation + EDTA; (C) trypsin activation alone; (D) trypsin activation + Sulforaphane (2 μg/ml); (E) Trypsin activation + Sulforaphane (5 μg/ml).
aphane at a concentration of 5 μg/ml significantly reduced (1662.66 ± 117.72 cpm, 65.66%) the proliferation of B16F-10 melanoma cells. Considerable inhibition of proliferation was also observed when Sulforaphane was administered at a concentration of 2 μg/ml (2840.66 ± 27.57 cpm, 41.33%) and 1 μg/ml (3316.66 ± 117.55 cpm, 31.5%) (Table 5). Gelatin zymographic analysis As shown in Fig. 3 Sulforaphane inhibited the activation of matrix metalloproteinases produced by B16F-10 melanoma cells. Conditioned medium after trypsin activation showed digested clear areas at 92 and 72 kD which was identical to MMP-9 and MMP-2 activity (Fig. 3C). Gels loaded with conditioned medium without trypsin activation, did not show any clear degradative areas, indicating the inactive form of the enzyme (Fig. 3A). Trypsin activated conditioned medium loaded gels, when incubated with 10 mM EDTA did not show clear degradative areas which indicates that the enzyme responsible for degradation is metalloproteinase (Fig. 3B). When conditioned medium was treated with Sulforaphane during trypsin activation, no clear bands were observed (Fig. 3D and E) indicating that Sulforaphane inhibited the activation of procollagenase to active collagenase at concentrations of 2 and 5 μg/ml. Discussion In the present study, we analyzed the anti-metastatic activity of Sulforaphane and its mechanism of action. B16F-10 melanoma cells are highly metastatic and form colonies of tumour nodules in the lungs when administered through tail vein, which in turn promote lung fibrosis and collagen deposition.
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Lung collagen hydroxyproline content is a direct marker of lung fibrosis. During lung fibrosis, extracellular matrix, especially collagen is deposited massively in the alveoli of lungs. Fifteen to thirty percent of collagen is hydroxyproline and it results in defective pulmonary function. Administration of Sulforaphane by 3 different modalities–simultaneous, prophylactic and developed–resulted in significant reduction of hydroxyproline content, which in turn causes marked reduction in lung fibrosis. This was well in correlation with histopathological analysis and significant reduction of tumour nodules in sulforaphane treated animals. This may be the reason for increase in lifespan of Sulforaphane treated animals. The acidic modification of monosaccharides produced by the oxidation of CH2OH group to COOH group yield uronic acids where as basic modification yield amino sugars (hexosamines) and these form an integral part of many structural polysaccharides, and glycosaminoglycans found in the ECM. Hyaluronic acid (HA) is a GAG made of repeated disaccharide units of D-glucuronic acid and N-acetyl D-glucosamine (Tammi et al., 2002; Delpech et al., 1997) and is a component of tissue matrix and tissue fluid. Concentration of HA is elevated in several cancers regardless of the tumour grade (Hautmann et al., 2000; Setala et al., 1999) and supports tumour cell migration by interacting with cell surface HA receptors and there by promoting metastasis (Tammi et al., 2002; Delpech et al., 1997; Turley et al., 2002). Hyaluronidase degrades HA and liberates disaccharide units, which are good promoters of angiogenesis as well as modifiers of proliferation, adhesion and migration of endothelial cells (Rodden et al., 1989; West et al., 1985). This in turn will enhance the process of metastasis as well as tumour directed angiogenesis. In the presence of glucuronic acid lactone, which is an esterified form of glucuronic acid, prolyl hydroxylase enzyme converts prohydroxyproline to hydroxyproline (Voet and Voet, 1995), which is a direct marker of lung fibrosis. Hexosamine has an important role in the synthesis of N-acetyl neuraminic acid (sialic acid), which is a component of glycolipids present on the surface of tumour cells (Voet and Voet, 1995). The enhanced level of these monosaccharides in the control metastatic tumour bearing animals indicates the active growth and proliferation of tumour cells. Administration of Sulforaphane significantly reduced the uronic acid and hexosamine content in the tumour bearing animals. The inhibitory effect of Sulforaphane on invasion and proliferation of B16F-10 melanoma cells (in vitro) strongly support these results. Sialic acid, a family of acetylated derivatives of neuraminic acid, occurs as a terminal component at the non-reducing end of carbohydrate chains of glycoproteins and glycolipids. Neoplasm often have an increased concentration of sialic acid on the tumour cell surface and sialoglycoproteins are shed or secreted by some of these cells which increases their concentration in blood (Khadapkar et al., 1975; Kloppel et al., 1977). Sialylation is one of the most common and versatile type of terminal glycosylation (Schauer and Corfield, 1982; Leyon et al., 2005). Structural analysis of tumour associated carbohydrate antigens has shown that sialylated derivatives together with related structural changes are essential carbohydrate epitopes associated with malignant transformation (Holmes et al., 1986). In the
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present study, the increased sialic acid level in the control metastatic tumour bearing animals was significantly reduced in the animals treated with Sulforaphane. Gamma glutamyl transpeptidase (GGT) is a cellular proliferation marker and high level of GGT was found in the serum of control metastatic tumour bearing animals. GGT catalyses intracellular GSH break down and provide energy to the tumour cells by γ-glutamyl cycle (West et al., 1985). In rapidly dividing tumours, cysteine, whose concentration in blood is low, may become limiting for GSH synthesis and cell growth (Obrador et al., 2002; Hanigan, 1995). GGT ensures sufficient cysteine for GSH synthesis by an alternate pathway in which GGT cleaves extra cellular GSH releasing γ-glutamyl aminoacids and cysteinyl glycine, which is further cleaved by membrane bound dipeptidases into L-cysteine and glycine. Free γ-glutamyl aminoacids, L-cysteine and glycine are further transported into the cell (Meister, 1983). Administration of Sulforaphane significantly reduced the serum GGT level. Matrix metalloproteinases are a family of N20 zincdependent endoproteinases that are capable of degrading almost all of the components of the extracellular matrix and thereby up regulates invasion and metastasis (Stetler-stevenson et al., 1996; Chambers and Matrisian, 1997). Among the MMPs reported earlier, MMP-2 and MMP-9 are key enzymes for degrading type IV collagen, which is a major component of the basement membrane (Zucker et al., 1993; Bernhard et al., 1994). Several experiments also proved that MMPs not only break down the physical barrier of extracellular matrix but also modulates the growth factors and cytokines stored in the extracellular matrix, which may promote neoplastic progression (Voet and Voet, 1995). The result of zymographic analysis indicates that administration of Sulforaphane inhibited the activation of matrix metalloproteinases. It also inhibited the invasion of B16F-10 melanoma cells through the collagen matrix and strongly back up the direct inhibition of activation of matrix metalloproteinases by Sulforaphane. The results of the above experiments strongly suggest the antimetastatic activity of Sulforaphane and this may be mainly due to the inhibition of activation of matrix metalloproteinases. Acknowledgements Authors acknowledge Dr. Ramadasan Kuttan, Research Director, Amala Cancer Research Centre, for his kind support in this study. One of the authors (P. Thejass) acknowledges CSIR, India for financial support in the form of JRF. References Albini, A., Iwamoto, H.K., Kleinman, G.R., et al., 1987. A rapid in vitro assay quantitating the invasive potential of tumor cells. Cancer Research 47, 3239–3245. Bergman, I., Loxley, R., 1940. The determination of hydroxyproline in urinehydrolysate. Clinica Chimica Acta 27, 347–349. Bernhard, E.J., Gruber, S.B., Muschel, R.J., 1994. Direct evidence linking expression of matrix metalloproteinases 9 (92-Kda gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proceedings of the National Academy Science 91, 4293–4297.
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Antimetastatic activity of Sulforaphane P. Thejass, Girija Kuttan ⁎ Department of Immunology, Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala State 680555, India Received 3 September 2005; accepted 2 December 2005
Abstract The effect of Sulforaphane on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice by three different modalities of administration—simultaneous, prophylactic and after tumour developed. Of this simultaneous mode of Sulforaphane administration was found to be most effective. There was 95.5% inhibition of lung tumour nodule formation and 94.06% increase in the life span of metastatic tumour bearing animals. Highly elevated levels of lung hydroxyproline, lung uronic acid, lung hexosamine, serum sialic acid and serum γ-glutamyl transpeptidase (GGT) in the metastatic control animals was found to be significantly lowered in the Sulforaphane treated animals. Histopathological analysis of lung tissues also correlated with these results. In the in vitro system Sulforaphane showed a significant inhibition in the invasion of B16F-10 melanoma cells across the collagen matrix. 3H-thymidine proliferation assay showed that Sulforaphane could inhibit the proliferation of B16F-10 melanoma cells in vitro. Gelatin zymographic analysis showed that Sulforaphane could inhibit the activation of matrix metalloproteinases. These findings suggest that Sulforaphane reduced the invasion of B16F-10 melanoma cells by the inhibition of activation of matrix metalloproteinases, thereby inhibiting lung metastasis. © 2006 Published by Elsevier Inc. Keywords: B16F-10 melanoma cells; Invasion; Matrix metalloproteinases; Metastasis; Proliferation; Sulforaphane
Introduction Metastasis or secondary neoplastic growth defines malignancy and is a major contributor to cancer mortality. The prognosis of cancer is mainly determined by the invasiveness of the tumour and its ability to metastasize. The metastatic process consists of several sequential events including escape of cancer cells from the original tumour, intravasation and dissemination through blood and lymphatic vessels, arrest in the microvasculature of the target organs, extravasation and proliferation at a new site (Fidler, 1978; Nicolson, 1982; Liotta, 1984; Netland and Zetter, 1989). Since basement membrane and extracellular matrix provide the main physical barriers to cancer cell invasion; proteolytic degradation of these structures has been proposed to be important in the metastatic process (Liotta, 1986a,b; Liotta et al., 1980). Any drug, which can inhibit one of the steps in the cascade, will be useful in the inhibition of tumour metastasis. Although there are several drugs available to control cancer growth in humans, there are no drugs presently available ⁎ Corresponding author. Tel.: +91 487 2307950; fax: +91 487 2307868. E-mail address: [email protected] (G. Kuttan). 0024-3205/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.lfs.2005.12.038
to specifically inhibit the metastasis of cancer cells. This is due to the fact that the cancer cells in different metastases and even in single metastases may respond differently to radiotherapy or chemotherapy. Isothiocyanates, the enzymatic hydrolysis product of glucosinolates, are naturally occurring constituents of cruciferous vegetables. Many isothiocyanates protect against chemically induced tumours in a variety of animal organs (Zhang and Talalay, 1994; Hecht, 1995). Phenyl and benzyl isothiocyanates inhibited carcinogenesis induced by carcinogens such as diethyl nitrosamine, dimethyl benzo (a) anthracene or benzo (a) pyrene (Wattenberg, 1987). The antimetastatic activity of Allyl isothiocyanate (AITC), and Phenyl isothiocyanate (PITC) has already been reported from our laboratory (Manesh and Kuttan, 2003). Sulforaphane, an isothiocyanate rich in broccoli (Brassica oleracea), is an inducer of phase II enzymes, which enhances detoxification of carcinogens, a mechanism contributing to anticarcinogenic activity (Zhang et al., 1992). Experiments in animal models showed that Sulforaphane protected the mouse forestomach against the neoplastic effects of B (a) P (Fahey et al., 2002). In the present study, the effect of Sulforaphane on the
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inhibition of pulmonary metastasis induced by B16F-10 melanoma cells in C57BL/6 mice was evaluated with special emphasis on the mechanism of action using in vitro models. Materials and methods Animals
histopathological analysis. The rest of the six animals in each group were observed for their survival. The mortality of the animals was observed and the percentage increase in life span (%ILS) was calculated using the formula %ILS = T − C / C × 100, where T represents the number of survival days of treated animals and C represents the number of survival days of control animals.
C57BL/6 mice (20–25 g body wt, 6–8 weeks old males) were purchased from National Institute of Nutrition, Hyderabad, India. The animals were fed with mouse chow (Sai Feeds, India) and water ad libitum. All animal experiments were conducted according to the rules and regulations of the Animal Ethics Committee, Govt. of India.
Histopathological analysis Lung tissues were fixed in 10% formalin, dehydrated in different concentrations of alcohol and embedded in paraffin wax. Sections (4 μm) were stained with eosin and hematoxylin.
Cell line
Determination of the cytotoxic activity of Sulforaphane towards B16F-10 melanoma cells in culture by MTT assay B16F-10 melanoma cells were seeded (5000 cells/well) in 96-well flat bottomed titre plate and incubated for 24 h at 37 °C in 5% CO2 atmosphere. Different concentrations of Sulforaphane (1–50 μg/ml) were added and incubated further for 48 h. Before 4 h of completion of incubation, 20 μl MTT (5 mg/ml) was added (Cole, 1986; Campling et al., 1991). Percentage of dead cells was determined using an ELISA plate reader set to record absorbance at 570 nm.
B16F-10 melanoma, a highly metastatic cell line was obtained from the National Centre for Cell Sciences, Pune, India. The cells were maintained in DMEM, supplemented with 10% FCS and antibiotics. Chemicals Sulforaphane and hydroxyproline were purchased from Sigma Chemicals, St.Louis, MO, USA. N-acetyl neuraminic acid was purchased from Sisco Research Laboratory, Mumbai. All other reagents were of analytical grade. Drug administration Sulforaphane was suspended in phosphate buffered saline (PBS, pH-7.4) and used for all the experiments. Determination of the antimetastatic activity in the in vivo system Determination of the effect of Sulforaphane on lung tumour nodule formation and rate of survival C57BL/6 mice were divided into 4 groups (14 nos/group). All the animals were injected with B16F-10 melanoma cells (106 cells/ animal) through lateral tail vein. (Liotta, 1986a,b). To three groups of animals Sulforaphane was administered intraperitoneally at a dose of 500 μg/kg body wt for 10 consecutive days in three different modalities—simultaneously with metastatic tumour cells (Group I); 10 days prior to metastatic tumour induction (Group II) and 10 days after tumour induction (Group III). Group IV animals were kept as untreated metastatic tumour bearing control. Eight animals from each group were sacrificed on the 21st day after tumour challenge, lungs were excised and blood was collected. Lungs were used for morphological examinations of metastatic tumour nodules and for the estimation of collagen hydroxyproline (Bergman and Loxley, 1940), hexosamine (Elson and Morgan, 1933) and uronic acid (Bitter and Muir, 1962) contents. Serum was separated from the blood and used for determining the sialic acid (Skoza and Mohos, 1976) and γ-glutamyl transpeptidase (GGT) (Szasz, 1976) levels. A portion of the lung was used for
Determination of antimetastatic activity using in vitro models
Collagen matrix invasion assay The invasion assay was carried out in modified Boyden chambers as described by Albini et al (Albini et al., 1987). The lower compartment of the chamber was filled with serum free DMEM and a polycarbonate filter coated with 25 μg Type I collagen was placed above this. B16F-10 melanoma cells (105 cells/150 μl DMEM) were then seeded on to the upper chamber in the presence and absence of Sulforaphane (1, 2 and 5 μg/ml) and incubated at 37 °C in 5% CO2 atmosphere for 10 h. After incubation, the filters were removed, fixed with methanol and stained with crystal violet. Cells migrating to the lower surface of the polycarbonate filters were counted under a microscope. The results were expressed as percentage inhibition of invasion. Determination of the effect of Sulforaphane on the inhibition of tumour cell proliferation (3H-thymidine incorporation assay) B16F-10 melanoma cells (5000 cells/well) were plated in a 96-well culture plate and incubated at 37 °C in 5% CO2 atmosphere. After 24 h, various concentrations of Sulforaphane (1, 2 and 5 μg/ml) were added and further incubated for 48 h. 3 H-thymidine was added to each well (1 μCi /well) and incubation was continued for additional 18 h. After completing incubation, the plates were centrifuged and the culture supernatant was removed, the cells were washed three times with PBS and then treated with ice cold PCA for 15 min. The resulting precipitate was dissolved in 0.5 N NaOH and was added to the scintillation fluid and kept overnight in the dark.
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The radioactivity was counted using a Rack Beta liquid scintillation counter.
Table 2 Effect of Sulforaphane on the lung biochemical parameters of metastases bearing animals
Gelatin zymography
Treatment
Hydroxyproline (μg/mg protein)
Uronic acid (μg/100 mg tissue wet wt)
Hexosamine (mg/100 mg tissue dry wt)
Normal Control Sulforaphane (500 μg/kg body wt) Prophylactic Simultaneous Developed
1.19 ± 0.10 21.91 ± 1.16
32.20 ± 2.00 352.62 ± 8.33
0.40 ± 0.10 4.12 ± 0.29
8.95 ± 0.66⁎ 4.99 ± 0.35⁎ 14.66 ± 0.70⁎
112.93 ± 5.10⁎ 85.14 ± 6.09⁎ 162.81 ± 7.13⁎
1.39 ± 0.17⁎ 0.90 ± 0.12⁎ 2.09 ± 0.15⁎
SDS-PAGE was performed with 5% gelatin incorporated in the separating gel (Billings et al., 1991). B16F-10 melanoma cells of subconfluent cultures were incubated with serum free medium for 24 h at 37 °C in 5% CO2 atmosphere. The conditioned medium was then collected and subjected to zymographic analysis. Fifty microlitres of sample (equivalent to 100 μg protein) was activated with 5 μl trypsin solution (75 μg/ml) in the presence and absence of Sulforaphane (2 and 5μg/ml) in 0.1 M Tris–HCl, 10 mM CaCl2 buffer (pH-8.0) and incubated for 1 h at room temperature. Samples were mixed with an equal volume of 2 × sample buffer and loaded on to 11% polyacrylamide gels containing 5% gelatin. Electrophoresis was carried out at 4 °C with constant current of 2 mA/tube until the tracking dye reached the periphery. The gels were then washed with 2% Triton X-100 in 0.1 M Tris–HCl, 10 mM CaCl2 at 37 °C for 18 h followed by staining with Gelcode Blue stain reagent for 2 h. Gels were destained to visualize the clear area against the dark background. Statistical analysis Values were expressed as mean ± S.D. The statistical analysis was done by using one-way ANOVA followed by Dunnett's test. Results Determination of antimetastatic activity in the in vivo system
The lungs were dissected out and assayed different biochemical parameters on the 22nd day after induction of B16F-10 melanoma cells (106 cells) through the lateral tail vein. For Prophylactic group of animals, drug administration started 10 days prior to tumour challenge, simultaneous group received the drug simultaneously with tumour challenge and developed group were given drug, 10 days after tumour challenge. Values are mean ± S.D., ⁎p b 0.001.
1994). The three different modalities of compound administration were found to be significantly effective. Of this simultaneous mode of administration produced maximum inhibition of 95.5% followed by prophylactic mode of administration (90.5%) and administration after tumour development (82.5%). Administration of Sulforaphane significantly increased the life span of tumour bearing animals (Table 1). The life span was highly enhanced in the case of simultaneous mode of administration (94.06%). Prophylactic administration enhanced the life span by 62.17% where as administration after tumour development increased the lifespan by 37.85%. Effect of Sulforaphane on the biochemical parameters of the metastasis bearing animals
Effect of Sulforaphane on the lung tumour nodule formation Metastatic tumour bearing animals treated with Sulforaphane showed significant reduction in tumour nodule formation. (Table 1). Metastatic control animals had massive tumour growth and were assigned an arbitrary number of 250 (Hill et al., Table 1 Effect of Sulforaphane on lung colonization of B16F-10 melanoma cells and survival of animals Treatment
Number of lung tumour nodules
% inhibition of nodule formation
%ILS
Control Sulforaphane (500 μg/kg body wt) Prophylactic Simultaneous Developed
250 a
–
–
23.71 ± 4.11 ⁎ 11.25 ± 2.21 ⁎ 43.87 ± 6.85 ⁎
90.51 95.50 82.45
77.80 94.06 62.17
The lungs were dissected out and observed for metastases on the 22nd day after induction of B16F-10 melanoma cells (106 cells) through the lateral tail vein. Prophylactic group of animals received Sulforaphane prior to tumour induction (10 doses, i.p.), simultaneous group received the drug simultaneously with tumour induction where as developed group received drug after 10 days of tumour induction. Values are mean ± S.D. a An arbitrary number of 250 is given for massive number of tumour nodules. ⁎ p b 0.001.
Lung collagen hydroxyproline Effect of Sulforaphane on the lung biochemical parameters is presented in Table 2. Control metastatic tumour bearing animals showed an increased level of lung collagen hydroxyproline (21.91 ± 1.16 μg/mg protein), which was significantly reduced in animals treated with Sulforaphane by the simultaneous mode Table 3 Effect of Sulforaphane on serum sialic acid and GGT levels of B16F-10 melanoma bearing animals Treatment
Sialic acid (μg/mL serum)
GGT (nmol p-nitroaniline/mL serum)
Normal Control Sulforaphane (500 μg/kg body wt) Prophylactic Simultaneous Developed
21.30 ± 1.50 109.68 ± 1.67
24.00 ± 0.17 108.26 ± 2.16
59.51 ± 1.29 ⁎ 35.13 ± 0.90 ⁎ 92.88 ± 1.23 ⁎
54.93 ± 1.58 ⁎ 39.15 ± 1.13 ⁎ 76.58 ± 0.90 ⁎
The serum was collected on 22nd day of tumour challenge by B16F-10 melanoma cells through lateral tail vein and assayed for serum biochemical parameters. Values are mean ± S.D. ⁎ p b 0.001.
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of administration (4.99 ± 0.35 μg/mg protein). Prophylactic mode of administration was also found to be effective with a reduced level of lung collagen hydroxyproline content (8.95 ± 0.66 μg/mg protein). Treatment of Sulforaphane after tumour development also produced considerable reduction of lung collagen hydroxyproline (14.66 ± 0.7 μg/mg protein), but not as much as the other two modalities. Lung hexosamine content Lung hexosamine level was highly increased in control metastatic tumour bearing animals (4.12 ± 0.29 mg/100 mg tissue dry wt) compared to the normal animals (0.4 ± 0.1 mg/100 mg tissue dry wt). When Sulforaphane was administered simultaneously with the tumour cells this elevated level was reduced to 0.9 ±
0.12 mg/100 mg tissue dry wt and when it was administered prophylactically the level was 1.39 ± 0.17 mg/100 mg tissue dry wt. Administration of Sulforaphane after tumour development also reduced this level to 2.09 ± 0.15 mg/100 mg tissue dry wt (Table 2). Uronic acid levels of the lung In control metastatic tumour bearing animals, the lung uronic acid level was drastically elevated (352.62 ± 8.33 μg/100 mg tissue wet wt), as compared to normal level (32.2 ± 2 μg/100 mg tissue wet wt) which was significantly reduced after the simultaneous administration of Sulforaphane (85.14 ± 6.09 μg/100 mg tissue wet wt). Considerable reduction in the lung uronic acid level was also obtained after prophylactic (112.93 ± 5.1 μg/
Fig. 1. Histopathological analysis of lungs from metastatic tumour bearing mice. (a) Normal; (b) control; (c) treated with Sulforaphane by simultaneous treatment modality; (d) treated with Sulforaphane by prophylactic treatment modality; (e) treated with Sulforaphane after tumour development.
P. Thejass, G. Kuttan / Life Sciences 78 (2006) 3043–3050 Table 4 Cytotoxicity of Sulforaphane towards B16F-10 melanoma cells in culture Concentration (μg/ml) of Sulforaphane
Percentage of cytotoxicity
1 2 5 10 20 25 50
0.4 2.4 3.6 6.4 17.2 32.4 42
B16F-10 melanoma cells were incubated with different concentrations (1–50 μg/ml) of Sulforaphane. Percentage of cytotoxicity was determined using MTT assay.
100 mg tissue wet wt) and developed (162.81 ± 7.13 μg/100 mg tissue wet wt) modalities of administration when compared to control group (Table 2). Serum sialic acid level The effect of Sulforaphane on the serum biochemical parameters is presented in Table 3. The serum sialic acid level of control metastatic tumour bearing animals was highly increased (108.26 ± 1.92 μg/ml serum) as compared to normal (21.3 ± 1.5 μg/ml serum). Here also the simultaneous adminis-
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tration of Sulforaphane significantly reduced the elevated serum sialic acid level to 35.13 ± 0.9 μg/ml serum, followed by prophylactic modality (59.51 ± 1.2 μg/ml serum) and in developed modality it was reduced only to 92.88 ± 1.23 μg/ml serum. Serum γ-glutamyl transpeptidase (GGT) level Here again, the highly elevated level of GGT in the serum of control metastatic tumour bearing animals (108.26 ± 1.92 n mol P-nitroaniline/ml serum) as compared to normal animals (24 ± 0.17 n mol P-nitroaniline/ml serum) was significantly reduced by the simultaneous mode of administration of Sulforaphane (39.15 ± 1.13 n mol P-nitroaniline/ml serum), followed by prophylactic mode of administration (54.93 ± 1.58 n mol Pnitroaniline/ml serum) where as in animals treated after tumour development this level was 76.58 ± 0.9 n mol P-nitroaniline/ml serum (Table 3). Histopathological analysis of lungs The hematoxylin and eosin stained sections of lung tissues are shown in Fig. 1(× 100). The lungs of control metastatic tumour bearing animals (Fig. 1b) showed prominent tumour
Fig. 2. The effect of Sulforaphane on the collagen matrix invasion. (a) Untreated control; (b) treatment with Sulforaphane (1 μg/ml); (c) treatment with Sulforaphane (2 μg/ml); (d) treatment with Sulforaphane (5 μg/ml).
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nodules around terminal bronchiole. These tumour nodules are characterized by polygonal tumour cells with prominent nucleolus, intracellular melanin deposition and clear areas of necrosis. This massive infiltration of the neoplastic cells around the main bronchioles, which make alveolar passages indistinguishable, extended to the pleura. This together with fibrosis reduces alveolar space, which in turn leads to reduced vital capacity. Simultaneous administration of Sulforaphane showed significant reduction in tumour mass. Alveoli and pleura were tumour free, alveolar passage lined with healthy ciliated columnar epithelial cells (Fig. 1c) and almost similar to normal lung (Fig. 1a). Considerable reduction of tumour mass was also observed in both prophylactic and developed modalities of administration (Fig. 1d and e). Determination of antimetastatic activity in the in vitro system Cytotoxic activity of Sulforaphane towards B16F-10 melanoma cells by MTT assay Cytotoxicity of Sulforaphane towards B16F-10 melanoma cells in culture is shown in Table 4. At 1–5 μg/ml Sulforaphane was found to be non-toxic to B16F-10 melanoma cells and these concentrations were used for further in vitro experiments. Collagen matrix invasion assay Metastatic B16F-10 melanoma cells show highly invasive property through the collagen matrix. Very high numbers of cells were found in the lower surface of the polycarbonate membrane, but administration of Sulforaphane produced significant inhibition in the invasion of the collagen matrix by the tumour cells in a dose dependent manner. At a concentration of 5 μg/ml Sulforaphane significantly inhibited the invasion of B16F-10 melanoma cells by 92.72% where as at 2 μg/ml and 1 μg/ml the percent of inhibition of invasion was found to be 61.18% and 36.42%, respectively (Fig. 2). Effect of Sulforaphane on tumour cell proliferation Proliferation rate was determined by the 3H-thymidine incorporation by the DNA of B16F-10 melanoma cells. Thymidine incorporation is proportional to the potential of the cells to synthesize DNA. Proliferation was expressed as radioactive count per minute. Untreated B16F-10 cells had very high rate of proliferation (4842.33 ± 230.7 cpm). Administration of SulforTable 5 Effect of Sulforaphane on tumour cell proliferation Treatment
Radioactive count/minute (CPM)
% inhibition
Untreated control Sulforaphane 1 μg 2 μg 5 μg
4842.33 ± 230.70
–
3316.66 ± 117.55 2840.66 ± 27.57 1662.00 ± 117.72
31.50 41.33 65.66
B16F-10 melanoma cells (5 × 103 cells/well) were grown in 96 well flat bottom plate After 24 h various concentrations of Sulforaphane were added and incubation was continued for 48 h. After incubation, 3H-thymidine was added to each well (1 μ Ci /well) and incubation was continued for 18 h. Cells were lysed and radioactivity was counted by using Rack Beta liquid scintillation counter. Values are mean ± S.D.
Fig. 3. Effect of Sulforaphane on the activation of matrix metalloproteinases. (A) Conditioned medium without trypsin activation; (B) trypsin activation + EDTA; (C) trypsin activation alone; (D) trypsin activation + Sulforaphane (2 μg/ml); (E) Trypsin activation + Sulforaphane (5 μg/ml).
aphane at a concentration of 5 μg/ml significantly reduced (1662.66 ± 117.72 cpm, 65.66%) the proliferation of B16F-10 melanoma cells. Considerable inhibition of proliferation was also observed when Sulforaphane was administered at a concentration of 2 μg/ml (2840.66 ± 27.57 cpm, 41.33%) and 1 μg/ml (3316.66 ± 117.55 cpm, 31.5%) (Table 5). Gelatin zymographic analysis As shown in Fig. 3 Sulforaphane inhibited the activation of matrix metalloproteinases produced by B16F-10 melanoma cells. Conditioned medium after trypsin activation showed digested clear areas at 92 and 72 kD which was identical to MMP-9 and MMP-2 activity (Fig. 3C). Gels loaded with conditioned medium without trypsin activation, did not show any clear degradative areas, indicating the inactive form of the enzyme (Fig. 3A). Trypsin activated conditioned medium loaded gels, when incubated with 10 mM EDTA did not show clear degradative areas which indicates that the enzyme responsible for degradation is metalloproteinase (Fig. 3B). When conditioned medium was treated with Sulforaphane during trypsin activation, no clear bands were observed (Fig. 3D and E) indicating that Sulforaphane inhibited the activation of procollagenase to active collagenase at concentrations of 2 and 5 μg/ml. Discussion In the present study, we analyzed the anti-metastatic activity of Sulforaphane and its mechanism of action. B16F-10 melanoma cells are highly metastatic and form colonies of tumour nodules in the lungs when administered through tail vein, which in turn promote lung fibrosis and collagen deposition.
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Lung collagen hydroxyproline content is a direct marker of lung fibrosis. During lung fibrosis, extracellular matrix, especially collagen is deposited massively in the alveoli of lungs. Fifteen to thirty percent of collagen is hydroxyproline and it results in defective pulmonary function. Administration of Sulforaphane by 3 different modalities–simultaneous, prophylactic and developed–resulted in significant reduction of hydroxyproline content, which in turn causes marked reduction in lung fibrosis. This was well in correlation with histopathological analysis and significant reduction of tumour nodules in sulforaphane treated animals. This may be the reason for increase in lifespan of Sulforaphane treated animals. The acidic modification of monosaccharides produced by the oxidation of CH2OH group to COOH group yield uronic acids where as basic modification yield amino sugars (hexosamines) and these form an integral part of many structural polysaccharides, and glycosaminoglycans found in the ECM. Hyaluronic acid (HA) is a GAG made of repeated disaccharide units of D-glucuronic acid and N-acetyl D-glucosamine (Tammi et al., 2002; Delpech et al., 1997) and is a component of tissue matrix and tissue fluid. Concentration of HA is elevated in several cancers regardless of the tumour grade (Hautmann et al., 2000; Setala et al., 1999) and supports tumour cell migration by interacting with cell surface HA receptors and there by promoting metastasis (Tammi et al., 2002; Delpech et al., 1997; Turley et al., 2002). Hyaluronidase degrades HA and liberates disaccharide units, which are good promoters of angiogenesis as well as modifiers of proliferation, adhesion and migration of endothelial cells (Rodden et al., 1989; West et al., 1985). This in turn will enhance the process of metastasis as well as tumour directed angiogenesis. In the presence of glucuronic acid lactone, which is an esterified form of glucuronic acid, prolyl hydroxylase enzyme converts prohydroxyproline to hydroxyproline (Voet and Voet, 1995), which is a direct marker of lung fibrosis. Hexosamine has an important role in the synthesis of N-acetyl neuraminic acid (sialic acid), which is a component of glycolipids present on the surface of tumour cells (Voet and Voet, 1995). The enhanced level of these monosaccharides in the control metastatic tumour bearing animals indicates the active growth and proliferation of tumour cells. Administration of Sulforaphane significantly reduced the uronic acid and hexosamine content in the tumour bearing animals. The inhibitory effect of Sulforaphane on invasion and proliferation of B16F-10 melanoma cells (in vitro) strongly support these results. Sialic acid, a family of acetylated derivatives of neuraminic acid, occurs as a terminal component at the non-reducing end of carbohydrate chains of glycoproteins and glycolipids. Neoplasm often have an increased concentration of sialic acid on the tumour cell surface and sialoglycoproteins are shed or secreted by some of these cells which increases their concentration in blood (Khadapkar et al., 1975; Kloppel et al., 1977). Sialylation is one of the most common and versatile type of terminal glycosylation (Schauer and Corfield, 1982; Leyon et al., 2005). Structural analysis of tumour associated carbohydrate antigens has shown that sialylated derivatives together with related structural changes are essential carbohydrate epitopes associated with malignant transformation (Holmes et al., 1986). In the
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present study, the increased sialic acid level in the control metastatic tumour bearing animals was significantly reduced in the animals treated with Sulforaphane. Gamma glutamyl transpeptidase (GGT) is a cellular proliferation marker and high level of GGT was found in the serum of control metastatic tumour bearing animals. GGT catalyses intracellular GSH break down and provide energy to the tumour cells by γ-glutamyl cycle (West et al., 1985). In rapidly dividing tumours, cysteine, whose concentration in blood is low, may become limiting for GSH synthesis and cell growth (Obrador et al., 2002; Hanigan, 1995). GGT ensures sufficient cysteine for GSH synthesis by an alternate pathway in which GGT cleaves extra cellular GSH releasing γ-glutamyl aminoacids and cysteinyl glycine, which is further cleaved by membrane bound dipeptidases into L-cysteine and glycine. Free γ-glutamyl aminoacids, L-cysteine and glycine are further transported into the cell (Meister, 1983). Administration of Sulforaphane significantly reduced the serum GGT level. Matrix metalloproteinases are a family of N20 zincdependent endoproteinases that are capable of degrading almost all of the components of the extracellular matrix and thereby up regulates invasion and metastasis (Stetler-stevenson et al., 1996; Chambers and Matrisian, 1997). Among the MMPs reported earlier, MMP-2 and MMP-9 are key enzymes for degrading type IV collagen, which is a major component of the basement membrane (Zucker et al., 1993; Bernhard et al., 1994). Several experiments also proved that MMPs not only break down the physical barrier of extracellular matrix but also modulates the growth factors and cytokines stored in the extracellular matrix, which may promote neoplastic progression (Voet and Voet, 1995). The result of zymographic analysis indicates that administration of Sulforaphane inhibited the activation of matrix metalloproteinases. It also inhibited the invasion of B16F-10 melanoma cells through the collagen matrix and strongly back up the direct inhibition of activation of matrix metalloproteinases by Sulforaphane. The results of the above experiments strongly suggest the antimetastatic activity of Sulforaphane and this may be mainly due to the inhibition of activation of matrix metalloproteinases. Acknowledgements Authors acknowledge Dr. Ramadasan Kuttan, Research Director, Amala Cancer Research Centre, for his kind support in this study. One of the authors (P. Thejass) acknowledges CSIR, India for financial support in the form of JRF. References Albini, A., Iwamoto, H.K., Kleinman, G.R., et al., 1987. A rapid in vitro assay quantitating the invasive potential of tumor cells. Cancer Research 47, 3239–3245. Bergman, I., Loxley, R., 1940. The determination of hydroxyproline in urinehydrolysate. Clinica Chimica Acta 27, 347–349. Bernhard, E.J., Gruber, S.B., Muschel, R.J., 1994. Direct evidence linking expression of matrix metalloproteinases 9 (92-Kda gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proceedings of the National Academy Science 91, 4293–4297.
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