Antimetastatic Activities of Selaginella Tamariscina (Beauv.) on Lung Cancer Cells in Vitro and in Vivo

Journal of Ethnopharmacology 110 (2007) 483–489

Antimetastatic activities of Selaginella tamariscina (Beauv.) on lung cancer cells in vitro and in vivo Shun Fa Yang a, Shu Chen Chu b, Shang Jung Liu c, Yi Chen Chen d, Yan Zin Chang e, Yih Shou Hsieh c,∗ a Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan b Department of Food Science, Central Taiwan University of Science and Technology, Taichung 406, Taiwan c Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan d School of Nutrition, Chung Shan Medical University, Taichung 402, Taiwan e Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung 402, Taiwan Received 18 May 2006; received in revised form 27 September 2006; accepted 12 October 2006 Available online 20 October 2006

Abstract Selaginella tamariscina is a traditional Chinese herb for the therapy of chronic trachitis and has been approved some anti-tumor activity. However, the anti-metastasis effects of Selaginella tamariscina in the lung cancer have not been understood clearly. The objectives of study were to investigate the effects of the Selaginella tamariscina extracts (STE) on the invasion and motility of highly metastatic A549 and Lewis lung carcinoma (LLC) cells. To further investigate the precise involvement of STE in tumor metastasis, A549 and LLC cells were treated with STE at various concentrations (0–100 ␮g/mL) for a speciﬁed period. The results from zymography showed that a STE treatment decreased (p < 0.05) the expressions of matrix metalloproteinase (MMP)-2, -9 and urokinase plasminogen activator (u-PA) in a dose-dependent manner in the A549 and LLC cell. Meanwhile, their endogenous inhibitors, which are tissue inhibitor of metalloproteinase-2 (TIMP-2) and plasminogen activator inhibitor-1 (PAI-1), were increased in the A549 cell. Furthermore, the inhibitory effect of STE on the growth and metastasis of LLC cells in vivo was also proven. These results demonstrated that STE could be a candidate antimetastatic agent against lung cancer. © 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Selaginella tamariscina; Invasion; Motility; MMP-2; u-PA

1. Introduction Selaginella tamariscina were demonstrated via an expression of p53 tumor suppressor gene and an induction of G1 arrest in the Selaginella tamariscina (Beauv.) is a traditional Chinese cell cycle to against human tumor cell lines (Lee et al., 1999). herb for the therapy of chronic trachitis, thrombocytopenic pur- Cancer metastasis, the spread of cancer cells from the primary pura and several forms of cancers in the orient (Zheng et al., neoplasm to distant sites and their growth there, is the major 1998; Dai et al., 2005). The major constituents in Selaginella cause of death in various cancer patients (Weiss, 1990). Metas- tamariscina are flavonoids (e.g. amentoflavone, hinokiflavone, tasis of cancer cells involves multiple processes and various sotetsuflavone and apogenin) (Cheong et al., 1998; Zheng cytophysiological changes, including changing adhesion capa- et al., 1998) and saccharides (e.g. trehalose, d-glucose, d- bility between cells and extracellular matrix (ECM) and dam- fructose and d-rhamnose) (Shimada et al., 1984). The crude aging intercellular interaction. Thus, a degradation of the ECM extracts of Selaginella tamariscina were evidenced to reduce and components of the basement membrane caused by a con- the gene expression and proinflammatory cytokines, includ- certed action of proteinases, such as matrix metalloproteinases ing interleukin-1␤ and TNF-␣ production in human mesangial (MMPs), cathepsins, and plasminogen activator (PA), play a cells (Kuo et al., 1998). In addition, the tumoricidal effects of critical role in tumor invasion and metastasis (Westermarck and Kahari, 1999; Yoon et al., 2003). Among these enzymes, MMP-2, MMP-9 and urokinase-PA (u-PA) could degrade most ∗ Corresponding author. Tel.: +886 4 24730022x11678; fax: +886 4 23248195. components of the ECM directly and deeply involved in cancer E-mail address: [email protected] (Y.S. Hsieh). invasion and metastasis (Bjorklund and Koivunen, 2005; Yang

Fig. 1. The chemical proﬁle of STE was analyzed by HPLC-mass spectrometer. (A) Chromatographic patterns from HPLC analysis (254 nm) of STE extracts showed peaks corresponding to the retention times (min). (B) The main product peak (with a retention time of 21.3 min as shown in (A)) was then subjected to mass spectrometer.

et al., 2005a). Therefore, the inhibition of migration or invasion achieved by increasing the concentration of solution B (100% mediated by MMP-2, -9 or u-PA could be a preventive way of acetonitrile) from 0 to 100% in 30 min at a flow rate of 1 ml/min. cancer metastasis (Bjorklund and Koivunen, 2005). Absorbance was monitored at 254 nm. The molecular masses Some pharmacological studies have demonstrated that of the peaks were determined from electrospray ionisation mass Selaginella tamariscina possesses anti-bacterial, anti- spectra using multiply-charged ion profile based on the modified hypertensive, anti-hyperglycemic (Miao et al., 1966; Zheng et method of Chang et al. (2005). For subsequent experiments, the al., 1998) and anti-tumoral activities (Lee et al., 1999). Based STE powder was dissolved in distilled water to achieve designed on our knowledge, the antimetastatic activities of Selaginella concentrations (0, 10, 25, 50, 75, and 100 ␮g/mL). tamariscina were not well studied previously. Therefore, in this study, the antimetastatic activities of STE on the cell 2.2. Cell and cell culture invasion were examined in vitro on A549, a highly metastatic human lung cancer cells. An in vivo study of LLC (Lewis lung A549 and LLC cell lines obtained from ATCC (Manas- carcinoma)-bearing mice, was also conducted to understand the sas, VA, USA) were cultured in Dulbecco’s modified Eagle’s antimetastatic activities of STE. medium (Life Technologies, Grand Island, NY, USA) sup- plemented with 10% fetal bovine serum, 2 mM glutamine, ␮ 2. Materials and methods 100 U/mL penicillin, 100 g/mL streptomycin, 0.1 mM non- essential amino acid and 1mM sodium pyruvate (Sigma chemical Co., St. Louis, MO, USA). All cell cultures were maintained 2.1. Preparation of Selaginella tamariscina extracts (STE) ◦ at 37 C in a humidified atmosphere of 5% CO2. Selaginella tamariscina (Beauv.) leaves were purchased from local herb stores in Taichung, Taiwan and the Selaginella 2.3. Determination of cell viability (MTT Assay) tamariscina extracts (STE) were prepared as described previously (Kuo et al., 1998). Briefly, 100 g of air-dried leaves To evaluate the cytotoxicity of STE, a MTT colorimetric were boiled at 70 ◦C for 24 h with 500 mL of 50% ethanol. assay was performed to determine the cell viability (Mosmann, The extraction procedure was repeated twice. Then, solvent was 1983). Cells were seeded in 24-well plates at a density of 4 removed from the combined extract with a vacuum rotary evap- 3.5 × 10 cells per well and treated with 0, 10, 25, 50, 75, and ◦ orator. The filtrate was then lyophilized and stored at −20 ◦C 100 ␮g/mL of STE at 37 C for 24 h. At the end of the expo- until further studies. A voucher specimen is deposited in The sure period, cells were washed with PBS and then incubated National Research Institute of Chinese Medicine, Taiwan (Kuo with 20 ␮L MTT (5 mg/mL) (Sigma chemical Co., St. Louis, et al., 1998). Furthermore, the extraction yield was 2.8% (w/w) MO, USA) for 4 h. The viable cell number is directly propor- and the chemical profile of Selaginella tamariscina extracts tional to the production of formazan following solublization with was analyzed by using high-pressure liquid chromatograms isopropanol, which can be measured spectrophotometrically at (HPLC)-mass spectrometer and shown in Fig. 1. Briefly, STE 563 nm (Beckman Spectrophotometer DU 640, Beckman Instru- were analyzed by HPLC-mass spectrometer using a HPLC ments, Fullerton, CA, USA). (Hitachi L-6200 with an L-4500 Diode Array detector) with a PE Sciex Qstar Pulsar ESI-TOF mass spectrometer. Samples 2.4. Cell invasion and motility assays (10 ␮l) were injected onto a Merck LiChrospher 100 RP-18 column (4 mm × 250 mm). The column was equilibrated in 0.05% The cell invasion and motility were assayed according to acetic acid/water (solution A) and elution of the components was the methods described by Chu et al. (2004). After being treated S.F. Yang et al. / Journal of Ethnopharmacology 110 (2007) 483–489 485 with STE for 24 h, surviving cells were harvested and seeded cells were scraped followed by a vortex at 4 ◦C for 10 min. Cell to Boyden chamber (Neuro Probe, Cabin John, MD, USA) at lysates were applied to a centrifugation of 10 000 × g at 4 ◦C for 104 cells per well in a serum free medium and then incubated for 10 min. Samples of cell lysates were separated in a 12.5% poly- 24hat37◦C. For invasion assay, 10 ␮L Matrigel (25 mg/50 mL; acrylamide gel and transferred onto a nitrocellulose membrane. BD Biosciences, MA, USA) was applied to 8 ␮m pore size The blot was subsequently incubated with 5% non-fat milk in polycarbonate membrane filters and the bottom chamber con- PBS for 1 h to block non-specific binding, with a polyclonal anti- tained standard medium. Filters were then air-dried for 5 h in a body against TIMP-2 (Serotec, Oxford, UK) or PAI-1(American laminar flow hood. The invaded cells were fixed with methanol Diagnostics Inc, Greenwich, CT, USA). The protein expres- and stained with Giemsa. Cell numbers were counted under sion was detected using an ECL plus detection kits (Amersham a light microscope. To determine the cell motility, cells were Life Sciences, Inc., Piscataway, NJ, USA). The relative photo- seeded into Boyden chamber on membrane filters which were graphic density was quantitated by scanning the photographic not coated with Matrigel. Migration of cells treated or untreated negatives on a gel documentation and analysis system (AlphaIm- with STE was measured as described in the invasion assay. ager 2000, Alpha Innotech Corporation, San Leandro, CA, USA). 2.5. Determination of MMP-2, -9 and u-PA by zymography 2.7. Measurement of lung metastasis and tumor growth in The activities of MMP-2 and MMP-9 in the medium were LLC-bearing mice measured by gelatin zymography protease assays as previously described (Yang et al., 2005b). Briefly, collected media of an C57BL/6 male mice (National Taiwan University Animal appropriate volume (adjusted by vital cell number) were pre- Center, Taiwan), with an age of 6-week-old, were implanted with pared with SDS sample buffer without boiling or reduction and 2 × 106 LLC cells (0.1 ml/mouse) via a subcutaneous injection. subjected to 0.1% gelatin–8% SDS-PAGE electrophoresis. After On the following day (Day 1), mice were randomly divided into electrophoresis, gels were washed with 2.5% Triton X-100 and four groups (n = 5 for each group). Mice were fed by an oral gav- then incubated in a reaction buffer (40 mM Tris–HCl, pH 8.0; age with water (control), STE (1 and 3 g/day/kg of body weight, ◦ 10 mM CaCl2 and 0.01% NaN3)at37 C for 12 h. Then the gel respectively) and doxorubicin (5 mg/day/kg of body weight). was stained with Coomassie brilliant blue R-250. The growth of tumors was determined every 3 days by direct Visualization of u-PA activity was performed by the method measurement with a calipers, and tumor volume was calculated described previously (Chu et al., 2004). Briefly, 2% casein (w/v) as the length × width × height (Kimura et al., 2003). After 30 and 20 ␮g/mL plasminogen were added to 8% SDS-PAGE gel. days, animals were euthanized, and the primary tumors were The u-PA activities of cells were performed as described in the isolated and weighed. Using a blunt-end 18-gauge needle, the gelatin zymography. lungs were filled with 1 mL of 5% India ink through the tra- chea. The lungs were isolated and the metastatic nodules on 2.6. Western blot analysis for determining TIMP-2 and the surface of lungs were counted with a black-stained back- PAI-1 protein levels ground under a microscopy (Sigounas et al., 2004; Chen et al., 2006). The protein levels of TIMP-2 and PAI-1 were measured by Western blot assays as previously described (Yang et al., 2.8. Statistical analysis 2003). Briefly, after an indicated STE treatment, the medium was removed and cells were rinsed with PBS twice. After the All statistical analyses were performed by using Student’s addition of 0.1 mL of cold RIPA buffer and protease inhibitors, t-test (Sigma-Stat 2.0 Jandel Scientific, San Rafael, CA, USA).

Table 1 Effects of various STE concentrations on the cell viability, cell invasion and motility of A549 and LLC cells Cell viability (% of control) Cell invasion (% of control) Cell motility (% of control)

STE concentrations in A549 cell 10 ␮g/mL 101.91 ± 1.51 98.41 ± 2.86 97.29 ± 3.12 25 ␮g/mL 97.89 ± 3.93 73.81 ± 3.63** 76.58 ± 3.60** 50 ␮g/mL 96.21 ± 2.39 48.41 ± 4.19*** 49.55 ± 2.38*** 75 ␮g/mL 96.53 ± 2.19 42.86 ± 3.63*** 43.24 ± 4.13*** 100 ␮g/mL 94.13 ± 1.86 38.89 ± 5.20*** 38.74 ± 5.01*** STE concentrations in LLC cell 10 ␮g/mL 65.24 ± 1.48*** 91.98 ± 2.95 90.36 ± 4.51 25 ␮g/mL 56.96 ± 3.01*** 63.68 ± 4.09** 68.53 ± 4.39** 50 ␮g/mL 44.01 ± 1.98*** 30.19 ± 2.87*** 29.44 ± 3.66*** 75 ␮g/mL 39.09 ± 1.67*** 20.75 ± 2.06*** 18.78 ± 2.21*** 100 ␮g/mL 22.86 ± 0.62*** 10.85 ± 1.25*** 11.17 ± 1.83***

Data represent the mean ± S.D. of at least three independent experiments (**p < 0.01, ***p < 0.001). 486 S.F. Yang et al. / Journal of Ethnopharmacology 110 (2007) 483–489

Fig. 2. Effects of STE on the activities of MMP-2, MMP-9 and u-PA. A549 cells and LLC cells were treated with 0, 10, 25, 50, 75 or 100 ␮g/mL of STE for 24 h and then subjected to gelatin and casein zymography to analyze the activities of MMP-2 and u-PA (A) on the A549 cells, MMP-9 and u-PA (B) on the LLC cells, respectively. Activities of these proteins were determined by a densitometric analysis with that of control as 100% (**p < 0.01; ***p < 0.001).

A signiﬁcant difference was used at 0.05 probability level. For concentration-dependent manner on A549 and LLC cell, as IC50 determination, the trend line of cell viability (MTT Assay) shown in Fig. 2A and B, respectively. Furthermore, the reduc- was obtained by using regression analysis (Sigma-Stat 2.0 Jandel tions (p < 0.05) on u-PA activities of A549 and LLC cells were Scientiﬁc, San Rafael, CA, USA). also measured when STE treated concentrations were beyond 50 ␮g/mL (Fig. 2A and B). There were 10.9% and 18.5% of 3. Results u-PA activity in A549 and LLC cells after a 100 ␮g/mL STE treatment when compared with those of non-STE treated group. 3.1. Effect of STE on the viability of A549 and LLC cells

The cytotoxic effect of various concentrations of STE 3.4. Inhibitory effects of STE on the protein levels of (0–100 ␮g/mL) on A549 and LLC cells were shown in Table 1. TIMP-2 and PAI-1 Although STE treatments had no (p > 0.05) cytotoxic effects on A549 cells, the viability of LLC cancer cells was decreased The Western blot was applied to analyze effects of STE on (p < 0.05) by STE treatments in a dose-dependent manner, with TIMP-2 and PAI-1 expression. Physiological activities of MMP- an IC50 of approximate 42.5 ␮g/mL. 2 and u-PA were related to levels of the speciﬁc endogenous inhibitors, TIMP-2 and PAI-1. The TIMP-2 and PAI-1 protein 3.2. Inhibition on invasion and motility of A549 and LLC levels in A549 cells were increased in a concentration-dependent cells by STE manner (Fig. 3A) (p < 0.05) while LLC cells were no changed (Fig. 3B) in STE treated groups compared with those of non-STE Using a cell invasion and motility assay with Boyden cham- treated group. ber, the reductions (p < 0.05) on invasion and motility of A549 cells were observed when the STE concentrations were beyond 3.5. The anti-metastatic effects of STE in vivo 25 ␮g/mL (Table 1). In the 100 ␮g/mL STE treated group, the invasion and motility of A549 cells showed 38.9% and 38.7%, To verify the in vivo anti-metastatic effects of STE, LLC- respectively, compared with those of non-STE treated groups. bearing C57BL/6 mice were treated with different concentra- Similar results were also obtained in LLC cells (Table 1). tion of STE. Furthermore, doxorubicin (5 mg/day/kg of body 3.3. Inhibitory effects of STE on matrix metalloproteinase weight) was administered as positive control in this study. Lung and u-PA activity metastases of animals treated with higher concentration of STE (3 g/day/kg of body weight) have decreased (p < 0.05) by 72% Via gelatin zymography assays, it was shown that compared with that of the control group (Fig. 4A). Small solid STE reduced (p < 0.05) MMP-2 and MMP-9 activity in a tumors were observed on 5 days after the cell inoculation. A S.F. Yang et al. / Journal of Ethnopharmacology 110 (2007) 483–489 487

Fig. 3. Effects of STE on the protein levels of the endogenous inhibitors TIMP-2 and PAI-1. A549 cells (A) and LLC cells (B) were treated with 0, 10, 25, 50,75 or 100 ␮g/mL of STE for 24 h, and then cell lysates were subjected to SDS-PAGE followed by Western blotting with anti-TIMP-2 or anti-PAI-1 antibodies. Signals of proteins were visualized with an ECL detection system. Activities of these proteins were determined by a densitometric analysis with that of control as 100% (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 4. The in vivo anti-metastasis effect of STE. After subcutaneous implantating LLC cells, C57BL/6 mice were treated with STE and then analyzed for the number of lung metastasis (A), growth of tumor (B), weight of primary tumor (C), and body weight (D). Data represent the mean ± S.D. (n =5)(**p < 0.01; ***p < 0.001). 488 S.F. Yang et al. / Journal of Ethnopharmacology 110 (2007) 483–489

53% reduction in higher concentration of STE-treated animals are interdependent in tumor invasion, while tumor cells showed was recorded on Day 30, compared with that of non-STE treated only low levels of invasion in the absence of either u-PAor MMP- groups (p < 0.05) (Fig. 4B). Moreover, by Day 30, a higher con- 9(Kim et al., 1998). Furthermore, Itoh et al. (1999) reported that centration of STE treatment induced a 79% reduction (p < 0.05) metastatic colonies were seldom observed in MMP-9-deficient in tumor weight (Fig. 4C) without any apparent signs of toxicity mice injected intravenously with melanoma or lung carcinoma as evidenced by a body weight monitoring (Fig. 4D) throughout cells. The positive relationships between MMPs (i.e. MMP-2 the experiment. and MMP-9) and metastasis in patients with breast cancer have been reported (Duffy et al., 2000; Hanemaaijer et al., 2000). 4. Discussion Dumitrescu and Shields (2005) also revealed that MMP-2 activity is highly related with the risk for a relapse in breast can- Selaginella tamariscina is often used to treat chronic trachitis, cer patients. Therefore, inhibition of the invasion mediated by thrombocytopenic purpura and several forms of cancers (Zheng MMP-2, -9 and u-PA should reduce the cancer metastasis. et al., 1998; Yin et al., 2005). However, the restraining effect In addition to anti-metastasis activity in vitro, in vivo and involved mechanism to the cancer metastasis of Selaginella antimetastatic activities of STE on LLC-bearing mice was also tamariscina has not been understood clearly. In this study, we conducted. As shown in Fig. 4, LLC-bearing mice, well estab- revealed that Selaginella tamariscina extract (STE) could sig- lished animal model for metastasis (Sigounas et al., 2004; Chen nificantly inhibit the invasive ability of A549 and LLC cancer et al., 2006), were applied as an animal model. Our results indi- cells and proved the inhibitory effect of STE on lung cancer cated that STE reduce both of the tumor growth and lung metas- invasiveness. Furthermore, our findings showed that STE could tasis ability of LLC-bearing C57BL/6 mice. On the other hand, decrease protein levels of tumor metastasis-related proteins such it was suggested that STE might have an inhibitory potential for as MMP-2, -9, and u-PA. According to our literature search, this the cells invasion and metastasis of A549 cells in vivo without is the first scientific report related to the inhibitory effect of STE any apparent sign of toxicity as proved by MTT assay. More- on lung cancer invasiveness via decreasing productions of tumor over, a consisted preliminary data with LLC cells also supported metastasis-related proteins. the effects of STE on the tumor weight of A549 bearing-nude In the present study, we observed that STE could signifi- mice. Although STE has no effects in cancer cell growth in vitro, cantly inhibit the invasive ability of A549 and LLC cancer cells. decreased tumor weight observed in A549 bearing-nude mice Dietary flavonoids were demonstrated several beneficial biolog- caused by STE may be related to the anti-angiogenesis effect ical activities in various cell types, such as anti-inflammation, of STE via lowering the levels of MMPs and u-PA, which may anti-oxidation, anti-proliferation, and proapoptosis (Ross and lead to the slow growth of tumor, rather than cause the death of Kasum, 2002). Several flavonoid compounds have been iso- cancer cells. lated from Selaginella tamariscina such as amentoflavone, api- In conclusion, STE exerted an inhibitory effect on several genin, hinokiflavone, sotetsuflavone, etc. (Cheong et al., 1998; essential steps of metastasis, including cell invasion and motil- Zheng et al., 1998; Yang et al., 2006). Among those com- ity. In addition, STE could regulate the activities of invasion- pounds, amentoflavone could reduce the histamine release from associated proteinases and their natural inhibitors. As evidenced rat peritoneal mast cells (Bronner and Landry, 1985) and api- from above results, STE may be a powerful candidate to devel- genin reduced the antigen-IgE-mediated hexosaminidase release ope a preventive agent for cancer metastasis. as well as TNF-␣ and IL-4 production from RBL-2H3 cells (Cheong et al., 1998). Oozes et al. (1999) reported that proin- Acknowledgment flammatory signals are mediated by TNF-␣ via pathways of nuclear factor-␬B (NF-␬B) and Akt. Ruiz and Haller (2006) evidenced that apigenin inhibits the proinflammatory gene expres- This study was supported by a grant of Committee on Chi- sion in intestinal epithelial cells by blocking Akt phosphory- nese Medicine and Pharmacy, Department of Health, Republic lation/activity. 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