PHYTOTHERAPY RESEARCH Phytother. Res. 29: 1366–1372 (2015) Published online 24 June 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5389
Paris Saponin VII Inhibits the Migration and Invasion in Human A549 Lung Cancer Cells Lei Fan,1,4† Yuhua Li,2† Yang Sun,1† Jing Han,1 Zhenggang Yue,1 Jin Meng,3 Xutao Zhang,1 Feng Zhang1* and Qibing Mei1* 1 Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica of the State Administration of Traditional Chinese Medicine, Collaborative Innovation Center for Chinese Medicine in Qinba Mountains, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, Shaanxi, PR China 2 No. 422 Hospital of PLA, Zhanjiang 524005, Guangdong, PR China 3 Department of Pharmacy, No. 309 Hospital of PLA, Beijing 100009, PR China 4 Department of Pharmacy, No. 210 Hospital of PLA, Liaoning 116000, PR China
Metastasis is the main cause of death in lung cancer. Targeting the process of metastasis is a strategy to lung cancer treatment. Trillium tschonoskii Maxim., a traditional Chinese medicine, has been used for treatment of many diseases, including cancer. This study aims to determine the anti-metastatic effect of paris saponin VII (PS VII) which was extracted from T. tschonoskii Maxim. by using human lung cancer cell line A549 cells. Our results showed that PS VII could significantly suppress the viability as well as cell migration and invasion abilities of A549 cells in a concentration-dependent manner. PS VII reduced the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 by elevating the expression of TIMP1/2. These data indicated that PS VII could reduce the metastatic capability of A549 cells, probably through up-regulating the expression of TIMP1/2. These findings demonstrated a new therapeutic potential for PS VII in anti-metastatic therapy of lung cancer. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: paris saponin VII; anti-metastasis; lung cancer; TIMP1/2.
INTRODUCTION Lung cancer is one of the most common cancer in the world; the annual diagnosis rate of new cases is approximately 1.6 million (Siegel et al., 2012). Although the treatment methods for lung cancer have been improved, the mortality in lung cancer patients still remains high. The first-line treatment of lung cancer is surgical resection, but many diagnosed cases of lung cancer are in an advanced stage and it is almost incurable when metastasis occurs (Shivapurkar et al., 2003). Lung cancer cells are highly invasive; the inhibition of their invasion ability may be effective in the treatment of lung cancer. In order to get the initiatives in fighting against lung cancer, more effective and well-tolerated drugs are needed. Many researches have showed that numerous natural compounds act as cancer preventative and therapeutic agents, and many anti-cancer drugs are derived from natural plant species (Cragg and Newman, 2013; Reddy et al., 2003; Sarkar et al., 2009). Trillium tschonoskii Maxim., also named ‘a pearl on head’, is a perennial herb of Trilliaceae found in mid-western China (Li et al., 2005). It has been used for treating hypertension, headache, neurasthenia, giddiness, and cancer, and ameliorating pains by the ancients of China.
* Correspondence to: Qibing Mei, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, Shaanxi, PR China; Feng Zhang, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, Shaanxi, PR China. E-mail: [email protected]; [email protected] † These authors contributed equally to this work.
Copyright © 2015 John Wiley & Sons, Ltd.
In our previous study, we extracted a kind of steroidal saponins, namely, paris saponin VII (PS VII) from T. tschonoskii Maxim. and found that it could suppress the growth of colorectal cancer cells and a murine model of xenograft tumor through inhibiting Ras activity (Li et al., 2014). In this study, we investigated the growth inhibitory effect and the anti-metastasis activities of this compound in a lung cancer cell line A549. Our results showed that PS VII could dose-dependently inhibit the migration and invasion of human A549 lung cancer cells.
MATERIALS AND METHODS Plant material. Root and rhizome part of Trillium tschonoski Maxim. were collected from Qinba Moutains, Shaanxi Province, China. Botanical samples were previously taxonomically identified, and voucher specimens were deposited in the laboratory of Pharmacology at the School of Pharmacy, Fourth Military Medical University. Extraction and isolation. The dried root and rhizome of Trillium (20.0 kg) were extracted with 70% ethanol for three times. After removal of the solvent under reduced pressure, the ethanol extract was suspended in H2O and subjected to macroporous resin (D101, Sunresin New Materials Co. Ltd., Shaanxi, China) column chromatography and eluted with increasing amounts of ethanol (i.e. 0% ethanol, 20% ethanol, 60% ethanol, and 95% ethanol). The 60%-ethanol fraction was
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separated by a silica gel column (Qingdao Haiyang Chemical Group Corporation, Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), reverse phase C18 chromatography (SiliCycle Corporation, Quebec, Canada), and MHPLC chromatography; final purification was achieved by HPLC to get compound PS VII. Its chemical structure is presented in Fig. 1. PS VII was dissolved in DMSO at 1 M and stocked at 20 °C with aliquots.
to the wounded region were observed by Olympus CK-2 inverted microscope and photographed (100× magnification) at 0 and 24 h. The wound area was measured by the program Image J. The cell wound closure rate was calculated according to the equation: Wound closure % = [1 (wound area at Tt / wound area at T0)] × 100%, where Tt is the time after wounding and T0 is the time immediately after wounding. The experiments were performed in triplicate.
Materials and chemicals. Triton X-100, pyruvate, penicillin G, and streptomycin were obtained from Sigma Chemical. DMEM and fetal bovine serums were purchased from Corning. Anti-MMP-2, MMP-9, TIMP-1, TIMP-2, and E-cadherin antibodies were purchased from Santa Cruz Biotechnology. Matrigel was from BD Biosciences. Materials and chemicals for electrophoresis were obtained from Bio-Rad. All other chemicals were of analytical reagent grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Cell invasion determinations. Cell invasion was determined by Matrigel-coated transwell cell culture chambers (8-μM pore size) (Millipore, Billerica, MA, USA) as described previously (Hsu et al., 2007). Cells were kept for 24 h in serum-free-medium, and then were trypsinized and resuspended in serum-free DMEM medium and placed in the upper chamber of the transwell insert (5 × 104 cells/well) and incubated with different concentrations (0.5, 1.5, and 4.5 μM) of PSVII. DMEM medium containing 10% FBS was added to the lower chamber. The cells were incubated at 37 °C in the incubator supplemented with 5% CO2 for 24 h, non-invasive cells in the upper chamber were removed by wiping with a cotton swab, and invasive cells were fixed with 4% formaldehyde in PBS and were stained with 1% crystal violet in 2% ethanol. Cells in the lower surface of the filter were photographed under a light microscope (100× magnification). The inserts were washed with 33% acetic acid. Absorbance of washing buffer at 570 nm was determined for each well using a microplate reader. Cell-free inserts containing only medium had been included in duplicate throughout each experiment as OD background controls. Reported OD data represent average background-corrected values ± SD obtained from three independent experiments in duplicate.
Cell line and culture conditions. Human lung carcinoma cell lines A549 (ATCC, Rockville, MD, USA) were cultured in DMEM medium. For cell maintenance, the basal medium was supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin. The solvent control contains an equivalent amount of DMSO corresponding to the highest used concentration of PS VII. Cell viability assay. The effects of PS VII on cell viability were determined by the MTT assay. Cells (5000 cells per well) were incubated with or without PS VII in triplicate in a 96-well plate and incubated for 24 h and 48 h at 37 ° C. After incubation, 20-μL MTT solution (5 mg/mL) was added to each well and incubated for another 4 h at 37 ° C. The supernatants were aspirated carefully, and 200-μL DMSO was added, and then the plate was holding on vibrator for 20 s. The optical density of the cell suspension was measured at 490 nm using a microplate reader (Bio-Tek instruments Inc., Winooski, VT). Cell viability was presented as mean ± S.D. of four independent experiments. Wound-healing assay. Cells were seeded in 6-well plates at a density of 5 × 105 cells/well. Once the cells reached 90% confluence, a wound area was carefully created by scraping the cell monolayer with a sterile 200-μL pipette tip, from one end to the other end of the well. The detached cells were removed by washing with PBS. Then, the cells were incubated at different concentrations (0.5, 1.5, and 4.5 μM) of PS VII. Cells migrated
Figure 1. Chemical structure of paris saponin VII (PS VII). Copyright © 2015 John Wiley & Sons, Ltd.
Morphological studies. For scanning electron microscopy, cells were trypsinized and resuspended in serumfree DMEM medium, and placed on glass coverslips. After cell attachment, 1.5 μM of PS VII was added. Cells were fixed with 2.5% glutaraldehyde for 45 min in 0.2 M cacodylate buffer. After post-fixation in 1% osmium tetroxide for 30 min, coverslips were dehydrated through a graded series of ethanol and amyl acetate and critical point dried. The preparation was coated with gold–palladium and observed with an Autoscan Etec Corporation scanning electron microscope at 20-kV acceleration voltage. Gelatin zymography. The zymographic analysis was adapted from Surgucheva IG (Surgucheva et al., 2003). Cells (5 × 105 cells per well) in a serum free medium were incubated with PSVII (0.5, 1.5, and 4.5 μM) in a 24-well plate and incubated for 24 h at 37 °C. After incubation, conditional medium was harvested and then electrophoresed on 15% denaturing sodium dodecyl sulfate (SDS) polyacrylamide gels containing 1 mg/mL of gelatin (Sangon, Shanghai, China). Gels were washed twice in raising buffer (50 mM Tris–HCl; 5 mM CaCl2; 2.5% Tri-ton-X 100; 1 μM ZnCl2; 0.05% NaN3) for 1 h, and then incubated for 24 h at 37 °C in the above buffer without Triton-X 100 so that renaturation of enzyme could occur. Gels were stained with Coomassie blue R-250 and destained with 5% acetic acid containing Phytother. Res. 29: 1366–1372 (2015)
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10% methanol. Gelatinolytic activities were visualized as clear bands against a blue background.
Figure 2. Effects of PS VII on A549 cell viability. Cells were incubated with or without PS VII (0.5, 1.5, 5, 15, 45, and 135 μM) for 24 h and 48 h at 37 °C. After incubation, the effects of PS VII on cell viability were determined by MTT assay. Cell viability is presented as mean ± S.D. of three independent experiments. There was a significant effect of different doses of PS VII at the p < 0.05 level for the seven conditions at 24 h [F(6, 14) = 448.89, p = 0.000], or at 48 h [F(6, 14) = 505.76, p = 0.000]. Post hoc comparisons using the LSD test indicated that the mean score for the 1.5 μM (M = 91.33, SD = 2.08), 5 μM (M = 78.33, SD = 2.52), 15 μM (M = 61.00, SD = 2.65), 45 μM (M = 46.67, SD = 4.04), and 135 μM (M = 22.33, SD = 2.08) was significantly different than the control (M = 100.00, SD = 0.00). However, 0.5 μM (M = 97.00, SD = 1.00) did not significantly differ from control at 24 h; the mean score for the 0.5 μM (M = 63.00, SD = 3.00), 1.5 μM (M = 56.27, SD = 3.51), 5 μM (M = 34.33, SD = 2.52), 15 μM (M = 22.33, SD = 3.21), 45 μM (M = 17.00, SD = 2.00), and 135 μM (M = 7.00, SD = 1.00) was significantly different than the control (M = 100.00, SD = 0.00) at 48 h. **P < 0.01 vs control of 24h; ## P < 0.01 vs control of 48 h.
Western blotting. After being treated with PS VII (0.5, 1.5, and 4.5 μM) for 24 h, cells were washed twice with PBS and treated with extraction buffer (50 mM Tris– Cl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% deoxycholic acid). The cell extractions were collected and centrifuged at 10 000 ×g for 15 min at 4 °C, and the supernatants were collected as cell lysates. The cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were blocked with 5% (w/v) non-fat milk in PBS containing 0.05% Tween-20, and then blotted with primary antibody. Subsequently, the membranes were incubated with an appropriate secondary antibody (horseradish peroxidaseconjugated goat anti-mouse or anti-rabbit IgG). The immuno-detected proteins were then revealed by enhanced chemiluminescence. Statistics. All data were processed by SPSS 17.0. A oneway between subjects ANOVA was conducted to compare the effect of different doses of PSVII (0.5, 1.5, or 4.5 μM) on growth, migration, invasion, or expression of TIMP-1, 2 of A549 cells, followed by a post hoc multiple comparisons using Fisher’s least significant difference (LSD) t-test. Differences were considered significant at P < 0.05.
Figure 3. Effects of PS VII on cell migration. (A) Migration of cancer cell line A549 was assessed by wound-healing assay. Cells were cultured to nearly confluent cell monolayer. A scratch wound was created on the cell surface using a micropipette tip, and then PS VII (0.5, 1.5, and 4.5 μM) were added. The cultures were incubated at 37 °C and photographed with microscope at 0 h and 24 h. (B) The cell wound closure rate was measured at 24 h and was calculated according to the equation: Wound closure % = [1 (wound area at Tt / wound area at T0)] × 100%, where Tt is the time after wounding and T0 is the time immediately after wounding. The experiments were performed in triplicate. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions [F(3, 8) = 18.28, p = 0.001]; Post hoc comparisons using the LSD test indicated that the mean score for the 1.5 μM (M = 73.00, SD = 3.61) and 4.5 μM (M = 83.00, SD = 3.00) was significantly different than the control (M = 67.00, SD = 3.00). However, 0.5 μM (M = 66.00, SD = 3.00) did not significantly differ from control. **P < 0.01 vs control. Copyright © 2015 John Wiley & Sons, Ltd.
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RESULTS PS VII inhibited cell viability of A549 cells The viability of A549 cells treated with PS VII at different concentrations (0.5, 1.5, 5, 15, 45, and 135 μM) for 24 h and 48 h was determined by MTT assay. As illustrated in Fig. 2, PS VII decreased cell viability rate in a concentration-dependent manner. A higher concentration of PS VII would inhibit the growth of A549 cells directly, then, in order to observe the effect of PS VII on migration and invasion of A549 cells more accurately and clearly, the concentrations of 0.5, 1.5, and 4.5 μM (showed no obvious cytotoxicity to cells proliferation) were chosen in the following steps. PS VII inhibited the migration of cells in vitro One characteristic of tumor metastasis is the increased ability of tumor cells migration. The inhibition of A549 cell migration by PS VII was investigated by wound-healing assays. As presented in Fig. 3A, after incubation with different concentrations of PS VII for 24 h, higher concentration led to significantly inhibition of cell migration (P < 0.01). The results are shown in Fig. 3B. PS VII inhibited A549 cell spreading Cell morphology was analyzed by scanning electron microscopy. As shown in Fig. 4, A549 cells treated with solvent were spreading out with long filopodia and flat lamellipodia. In contrast, some cells became round with little membrane protrusions after PS VII (1.5 μM) treatment. The number of cell filopodia and lamellipodia reduced significantly. PS VII inhibited invasion of A549 cells in vitro In order to determine the inhibitory effect of PS VII on the invasion of A549 cells across the extracellular matrix (ECM), the cells that invaded through Matrigel-coated polycarbonate filter in the transwell chamber were analyzed. The results are shown in Fig. 5. Fig. 5A showed that the majority of A549 cells in control group invaded from the upper chamber to the lower chamber. However, the penetration of the
Figure 5. Effect of PS VII on invasion of A549 cells. (A) Cells were treated with various concentrations of PS VII (0.5, 1.5, and 4.5 μM) for 24 h, and cell invasion assay was performed. The invaded cells were photographed (100 × magnification). (B) The invaded cells were quantified by the absorbance of the crystal violet washed with 33% acetic acid from the cells that invaded the underside of the porous polycarbonate membrane. The experiments were performed in triplicate and represented the average of three experiments ± S.D. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions [F(3, 8) = 107.04, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.69, SD = 0.05), 1.5 μM (M = 0.51, SD = 0.02), and 4.5 μM (M = 0.36, SD = 0.03) was significantly different than the control (M = 0.97, SD = 0.07). **P < 0.01 vs control.
Matrigel coated filter by A549 cells was inhibited in the presence of PS VII. The quantification of cells in the lower chamber from Fig. 5B indicated that PS VII significantly inhibited A549 cells invasion (P < 0.01), and this inhibitory effect was concentration dependent.
Figure 4. Effects of PS VII on cell spreading. Cell morphology was analyzed by scanning electron microscopy (1.5k×). Copyright © 2015 John Wiley & Sons, Ltd.
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PS VII suppressed activity of MMP-2 and MMP-9 To investigate MMP-2 and -9 activities in A549 cells, the conditioned medium was subjected to gelatin zymography. The gelatinolytic activity at 72 and 92 kDa, which corresponded to the molecular mass of MMP-2 and -9, was detected in the conditioned medium from cells treated with PS VII. Fig. 6 illustrated that the activity of MMP-2 and -9 was markedly repressed by PS VII in a concentrationdependent manner.
PS VII affected metastasis-related protein levels in A549 cells Because the enzyme activity of MMP-2 and -9 was markedly repressed by PS VII, the effect of PS VII on the expression of MMPs was investigated by Western blot. After incubation with different concentrations of PS VII for 24 h, PS VII did not suppress the expression of MMP-2 and -9 (data not shown). So we further detected the expression of TIMP-1 and TIMP-2, which are two regulatory factors of the enzyme activity of MMPs. Fig. 7 indicated the levels of TIMP-1 and TIMP-2 were up-regulated by PS VII (P < 0.01). Similarly, Fig. 8 showed the protein concentration of
Figure 6. Effect of PS VII on activations of MMP-2 and -9 in A549 cells. (A) Cells were incubated for 24 h with a vehicle control or PS VII (0.5, 1.5, and 4.5 μM). Conditioning media were used for the measurement of MMP-2 and MMP-9 enzyme activity levels by gelatin zymography as described in Materials and methods. (B) Densitometry showing different activities of MMP-2 and MMP-9 in medium samples is presented in A. The experiments were performed in triplicate and represented the average of three experiments ± S.D. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions for MMP-2 [F(3, 8) = 87.94, p = 0.000], or for MMP-9 [F(3, 8) = 117.17, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 9.23, SD = 1.10), 1.5 μM (M = 5.43, SD = 0.38), and 4.5 μM (M = 2.37, SD = 0.31) was significantly different than the control (M = 12.00, SD = 1.00) for MMP-2; Post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 25.33, SD = 1.53), 1.5 μM (M = 19.33, SD = 1.53), and 4.5 μM (M = 10.33, SD = 1.53) was significantly different than the control (M = 31.00, SD = 1.00) for MMP-9. **P < 0.01 vs control. Copyright © 2015 John Wiley & Sons, Ltd.
Figure 7. Effect of PS VII on the expression of TIMP-1, TIMP-2, and E-cadherin. (A) Cells were treated with PS VII for 24 h, and the protein expression levels were estimated by Western blot. (B) Determined levels of TIMP-1, TIMP-2, and E-cadherin were quantified by densitometric analysis. The densitometric results are expressed as mean ± S.D. of three independent experiments. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions for TIMP-1 [F(3, 8) = 105.66, p = 0.000], for TIMP-2 [F(3, 8) = 261.64, p = 0.000], or for E-cadherin [F(3, 8) = 382.24, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.29, SD = 0.05), 1.5 μM (M = 0.35, SD = 0.04), and 4.5 μM (M = 0.68, SD = 0.05) was significantly different than the control (M = 0.14, SD = 0.02) for TIMP-1. However, did not significantly differ from control; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.33, SD = 0.04), 1.5 μM (M = 0.83, SD = 0.05), and 4.5 μM (M = 0.93, SD = 0.04) was significantly different than the control (M = 0.17, SD = 0.04) for TIMP-2; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.66, SD = 0.03), 1.5 μM (M = 1.56, SD = 0.06), and 4.5 μM (M = 1.96, SD = 0.12) was significantly different than the control (M = 0.25, SD = 0.04) for E-cadherin. **P < 0.01 vs control.
TIMP-1 and TIMP-2 in the conditioned media was also up-regulated by PS VII (P < 0.01). These results suggest that PS VII may affect the activity of MMPs by elevating the expression of TIMPs, which in turn led to the inhibition of invasion. E-cadherin is one of the main epithelial adhesive molecules. Loss of E-cadherin is associated with metastasis Phytother. Res. 29: 1366–1372 (2015)
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Figure 8. Supernatant from cultured A549 cells was collected and used to determine secreted TIMP1/2 protein levels after the cells were treated with PS VII for 24 h by using a human TIMP1/2 enzyme-linked immunosorbent assay (ELISA). TIMP1/2 concentrations (ng/mL) were adjusted to the number of cultured cells. Error bars represent the S.D. from five independent experiments. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions for TIMP-1 [F(3, 8) = 40.89, p = 0.000], for TIMP-2 [F(3, 8) = 107.23, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 41.67, SD = 1.53), 1.5 μM (M = 46.33, SD = 1.53), and 4.5 μM (M = 51.33, SD = 2.52) was significantly different than the control (M = 34.33, SD = 2.08) for TIMP-1; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 59.33, SD = 1.53), 1.5 μM (M = 66.67, SD = 1.53), and 4.5 μM (M = 77.67, SD = 1.53) was significantly different than the control (M = 55.00, SD = 2.00) for TIMP-2. **P < 0.01 vs control.
and poor prognosis in many neoplasms (Buda and Pignatelli, 2011). We found that PS VII treatment could up-regulate E-cadherin levels effectively.
DISCUSSION Suppression of cancer metastasis is an urgent therapeutic need in treatment of lung cancer. But most existing drugs only inhibit cancer cell proliferation. Little has been accomplished in terms of treating cancer metastasis. In our previous study, we have found that PS VII, a saponin compound isolated from T. tschonoskii Maxim., has growth inhibitory effect in vitro as well as in vivo (Li et al., 2014). In addition, non-apoptotic processes, including inhibition of cancer metastasis (Yoon et al., 2012), were also involved in saponin cytotoxic activity. In the current study, we therefore investigated the anti-metastasis activity of PS VII using lung cancer A549 cells. Metastasis is a complex, multistep process. It includes migration, invasion, adhesion, infiltration, colonization at a distant site, and the subsequent formation of new microvessels (Weng and Yen, 2012). To successfully invade, cells must acquire the ability of migration. In a non-cytotoxic dose, we evaluated the anti-migratory activity of PS VII through the wound healing assay which was a classic in vitro assay of cell migration. Our results showed that PS VII could reduce cell migration rate in a concentration-dependent manner. Cell migration is a highly integrated multistep process, which is initiated by the formation of filopodia and lamellipodia in the cell membrane (Bailly and Condeelis, 2002). Copyright © 2015 John Wiley & Sons, Ltd.
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Migration of cells is based on cycles of lamellipodial extension, attachment, cell body translocation, and retraction of the cell (Berrier and Yamada, 2007). We found PS VII-treated A549 cells became round, with little membrane protrusions. The number of cell filopodia and lamellipodia reduced significantly, suggesting the motility of cancer cells were inhibited. Cell migration partly depends on efficiency of cell attachment and detachment between cells (Mitra et al., 2005). E-cadherin is an essential component of adherence junctions. E-cadherin binds with its extracellular domain to an E-cadherin molecule of the neighboring epithelial cell to stabilize cell-to-cell contacts. So we detected the expression of E-cadherin and the results showed that E-cadherin was up-regulated after the cells were treated with PS VII. This may be one of the reasons for the reduced cell migration rate. In this study, the invasive ability of cells was evaluated by transwell invasion assay. The results demonstrated that PS VII could inhibit cell invasion rate in a concentration-dependent manner. Cell invasion requires proteolysis of extracellular matrix (ECM) components. In these steps, expression of proteolytic enzymes, such as MMPs, is crucial for ECM degradation (Friedl and Wolf, 2003). Many studies have demonstrated that higher protein expression levels of MMP-2 and -9 were associated with a poorer prognosis (Lin et al., 2011). Therefore, we investigated whether the inhibitory effect of PS VII on cell invasion was through suppressing expression of MMP-2 and -9. Results from gelatin zymography showed that PS VII could suppress the proteolytic enzyme activity of MMP-2 and -9. However, the expression of these two proteins was not affected by PS VII. Activities of MMPs are controlled by their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs) such as TIMP-1 and TIMP-2 (Di Carlo, 2014), so we further detected the expression of TIMP-1 and -2. We found that TIMP-1 and -2 were up-regulated by PS VII treatment, suggesting that PS VII may affect the activity of MMPs by elevating the expression of TIMPs. Taken together, our data suggested that PS VII obtained from T. tschonoskii Maxim. could inhibit migration and invasion of A549 cells. The increase in the expression of TIMP-1/2 and E-cadherin may contribute to this effect. Base on our study, PS VII could be a promising agent for therapeutic and preventive purposes of lung cancer by not only suppressing the proliferation of cells but also inhibiting metastasis-associated events.
Acknowledgements We thank the Department of forestry of Shaanxi Province and the Taibaishan natural preserve of Shaanxi Province for helping us collect the Trillium tschonoskii Maxim. This investigation was supported by the grant from the Taibaishan Natural Preserve of Shaanxi Province, and the grant (No. 81302787) from the National Nature Science Foundation of China, and the grant (Nos. 2012M512102, 2013T60964) from the Postdoctoral Science Foundation of China.
Conflict of Interest The authors have declared that there is no conflict of interest. Phytother. Res. 29: 1366–1372 (2015)
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Phytother. Res. 29: 1366–1372 (2015)
Paris Saponin VII Inhibits the Migration and Invasion in Human A549 Lung Cancer Cells Lei Fan,1,4† Yuhua Li,2† Yang Sun,1† Jing Han,1 Zhenggang Yue,1 Jin Meng,3 Xutao Zhang,1 Feng Zhang1* and Qibing Mei1* 1 Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica of the State Administration of Traditional Chinese Medicine, Collaborative Innovation Center for Chinese Medicine in Qinba Mountains, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, Shaanxi, PR China 2 No. 422 Hospital of PLA, Zhanjiang 524005, Guangdong, PR China 3 Department of Pharmacy, No. 309 Hospital of PLA, Beijing 100009, PR China 4 Department of Pharmacy, No. 210 Hospital of PLA, Liaoning 116000, PR China
Metastasis is the main cause of death in lung cancer. Targeting the process of metastasis is a strategy to lung cancer treatment. Trillium tschonoskii Maxim., a traditional Chinese medicine, has been used for treatment of many diseases, including cancer. This study aims to determine the anti-metastatic effect of paris saponin VII (PS VII) which was extracted from T. tschonoskii Maxim. by using human lung cancer cell line A549 cells. Our results showed that PS VII could significantly suppress the viability as well as cell migration and invasion abilities of A549 cells in a concentration-dependent manner. PS VII reduced the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 by elevating the expression of TIMP1/2. These data indicated that PS VII could reduce the metastatic capability of A549 cells, probably through up-regulating the expression of TIMP1/2. These findings demonstrated a new therapeutic potential for PS VII in anti-metastatic therapy of lung cancer. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: paris saponin VII; anti-metastasis; lung cancer; TIMP1/2.
INTRODUCTION Lung cancer is one of the most common cancer in the world; the annual diagnosis rate of new cases is approximately 1.6 million (Siegel et al., 2012). Although the treatment methods for lung cancer have been improved, the mortality in lung cancer patients still remains high. The first-line treatment of lung cancer is surgical resection, but many diagnosed cases of lung cancer are in an advanced stage and it is almost incurable when metastasis occurs (Shivapurkar et al., 2003). Lung cancer cells are highly invasive; the inhibition of their invasion ability may be effective in the treatment of lung cancer. In order to get the initiatives in fighting against lung cancer, more effective and well-tolerated drugs are needed. Many researches have showed that numerous natural compounds act as cancer preventative and therapeutic agents, and many anti-cancer drugs are derived from natural plant species (Cragg and Newman, 2013; Reddy et al., 2003; Sarkar et al., 2009). Trillium tschonoskii Maxim., also named ‘a pearl on head’, is a perennial herb of Trilliaceae found in mid-western China (Li et al., 2005). It has been used for treating hypertension, headache, neurasthenia, giddiness, and cancer, and ameliorating pains by the ancients of China.
* Correspondence to: Qibing Mei, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, Shaanxi, PR China; Feng Zhang, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, Shaanxi, PR China. E-mail: [email protected]; [email protected] † These authors contributed equally to this work.
Copyright © 2015 John Wiley & Sons, Ltd.
In our previous study, we extracted a kind of steroidal saponins, namely, paris saponin VII (PS VII) from T. tschonoskii Maxim. and found that it could suppress the growth of colorectal cancer cells and a murine model of xenograft tumor through inhibiting Ras activity (Li et al., 2014). In this study, we investigated the growth inhibitory effect and the anti-metastasis activities of this compound in a lung cancer cell line A549. Our results showed that PS VII could dose-dependently inhibit the migration and invasion of human A549 lung cancer cells.
MATERIALS AND METHODS Plant material. Root and rhizome part of Trillium tschonoski Maxim. were collected from Qinba Moutains, Shaanxi Province, China. Botanical samples were previously taxonomically identified, and voucher specimens were deposited in the laboratory of Pharmacology at the School of Pharmacy, Fourth Military Medical University. Extraction and isolation. The dried root and rhizome of Trillium (20.0 kg) were extracted with 70% ethanol for three times. After removal of the solvent under reduced pressure, the ethanol extract was suspended in H2O and subjected to macroporous resin (D101, Sunresin New Materials Co. Ltd., Shaanxi, China) column chromatography and eluted with increasing amounts of ethanol (i.e. 0% ethanol, 20% ethanol, 60% ethanol, and 95% ethanol). The 60%-ethanol fraction was
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separated by a silica gel column (Qingdao Haiyang Chemical Group Corporation, Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), reverse phase C18 chromatography (SiliCycle Corporation, Quebec, Canada), and MHPLC chromatography; final purification was achieved by HPLC to get compound PS VII. Its chemical structure is presented in Fig. 1. PS VII was dissolved in DMSO at 1 M and stocked at 20 °C with aliquots.
to the wounded region were observed by Olympus CK-2 inverted microscope and photographed (100× magnification) at 0 and 24 h. The wound area was measured by the program Image J. The cell wound closure rate was calculated according to the equation: Wound closure % = [1 (wound area at Tt / wound area at T0)] × 100%, where Tt is the time after wounding and T0 is the time immediately after wounding. The experiments were performed in triplicate.
Materials and chemicals. Triton X-100, pyruvate, penicillin G, and streptomycin were obtained from Sigma Chemical. DMEM and fetal bovine serums were purchased from Corning. Anti-MMP-2, MMP-9, TIMP-1, TIMP-2, and E-cadherin antibodies were purchased from Santa Cruz Biotechnology. Matrigel was from BD Biosciences. Materials and chemicals for electrophoresis were obtained from Bio-Rad. All other chemicals were of analytical reagent grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Cell invasion determinations. Cell invasion was determined by Matrigel-coated transwell cell culture chambers (8-μM pore size) (Millipore, Billerica, MA, USA) as described previously (Hsu et al., 2007). Cells were kept for 24 h in serum-free-medium, and then were trypsinized and resuspended in serum-free DMEM medium and placed in the upper chamber of the transwell insert (5 × 104 cells/well) and incubated with different concentrations (0.5, 1.5, and 4.5 μM) of PSVII. DMEM medium containing 10% FBS was added to the lower chamber. The cells were incubated at 37 °C in the incubator supplemented with 5% CO2 for 24 h, non-invasive cells in the upper chamber were removed by wiping with a cotton swab, and invasive cells were fixed with 4% formaldehyde in PBS and were stained with 1% crystal violet in 2% ethanol. Cells in the lower surface of the filter were photographed under a light microscope (100× magnification). The inserts were washed with 33% acetic acid. Absorbance of washing buffer at 570 nm was determined for each well using a microplate reader. Cell-free inserts containing only medium had been included in duplicate throughout each experiment as OD background controls. Reported OD data represent average background-corrected values ± SD obtained from three independent experiments in duplicate.
Cell line and culture conditions. Human lung carcinoma cell lines A549 (ATCC, Rockville, MD, USA) were cultured in DMEM medium. For cell maintenance, the basal medium was supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin. The solvent control contains an equivalent amount of DMSO corresponding to the highest used concentration of PS VII. Cell viability assay. The effects of PS VII on cell viability were determined by the MTT assay. Cells (5000 cells per well) were incubated with or without PS VII in triplicate in a 96-well plate and incubated for 24 h and 48 h at 37 ° C. After incubation, 20-μL MTT solution (5 mg/mL) was added to each well and incubated for another 4 h at 37 ° C. The supernatants were aspirated carefully, and 200-μL DMSO was added, and then the plate was holding on vibrator for 20 s. The optical density of the cell suspension was measured at 490 nm using a microplate reader (Bio-Tek instruments Inc., Winooski, VT). Cell viability was presented as mean ± S.D. of four independent experiments. Wound-healing assay. Cells were seeded in 6-well plates at a density of 5 × 105 cells/well. Once the cells reached 90% confluence, a wound area was carefully created by scraping the cell monolayer with a sterile 200-μL pipette tip, from one end to the other end of the well. The detached cells were removed by washing with PBS. Then, the cells were incubated at different concentrations (0.5, 1.5, and 4.5 μM) of PS VII. Cells migrated
Figure 1. Chemical structure of paris saponin VII (PS VII). Copyright © 2015 John Wiley & Sons, Ltd.
Morphological studies. For scanning electron microscopy, cells were trypsinized and resuspended in serumfree DMEM medium, and placed on glass coverslips. After cell attachment, 1.5 μM of PS VII was added. Cells were fixed with 2.5% glutaraldehyde for 45 min in 0.2 M cacodylate buffer. After post-fixation in 1% osmium tetroxide for 30 min, coverslips were dehydrated through a graded series of ethanol and amyl acetate and critical point dried. The preparation was coated with gold–palladium and observed with an Autoscan Etec Corporation scanning electron microscope at 20-kV acceleration voltage. Gelatin zymography. The zymographic analysis was adapted from Surgucheva IG (Surgucheva et al., 2003). Cells (5 × 105 cells per well) in a serum free medium were incubated with PSVII (0.5, 1.5, and 4.5 μM) in a 24-well plate and incubated for 24 h at 37 °C. After incubation, conditional medium was harvested and then electrophoresed on 15% denaturing sodium dodecyl sulfate (SDS) polyacrylamide gels containing 1 mg/mL of gelatin (Sangon, Shanghai, China). Gels were washed twice in raising buffer (50 mM Tris–HCl; 5 mM CaCl2; 2.5% Tri-ton-X 100; 1 μM ZnCl2; 0.05% NaN3) for 1 h, and then incubated for 24 h at 37 °C in the above buffer without Triton-X 100 so that renaturation of enzyme could occur. Gels were stained with Coomassie blue R-250 and destained with 5% acetic acid containing Phytother. Res. 29: 1366–1372 (2015)
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10% methanol. Gelatinolytic activities were visualized as clear bands against a blue background.
Figure 2. Effects of PS VII on A549 cell viability. Cells were incubated with or without PS VII (0.5, 1.5, 5, 15, 45, and 135 μM) for 24 h and 48 h at 37 °C. After incubation, the effects of PS VII on cell viability were determined by MTT assay. Cell viability is presented as mean ± S.D. of three independent experiments. There was a significant effect of different doses of PS VII at the p < 0.05 level for the seven conditions at 24 h [F(6, 14) = 448.89, p = 0.000], or at 48 h [F(6, 14) = 505.76, p = 0.000]. Post hoc comparisons using the LSD test indicated that the mean score for the 1.5 μM (M = 91.33, SD = 2.08), 5 μM (M = 78.33, SD = 2.52), 15 μM (M = 61.00, SD = 2.65), 45 μM (M = 46.67, SD = 4.04), and 135 μM (M = 22.33, SD = 2.08) was significantly different than the control (M = 100.00, SD = 0.00). However, 0.5 μM (M = 97.00, SD = 1.00) did not significantly differ from control at 24 h; the mean score for the 0.5 μM (M = 63.00, SD = 3.00), 1.5 μM (M = 56.27, SD = 3.51), 5 μM (M = 34.33, SD = 2.52), 15 μM (M = 22.33, SD = 3.21), 45 μM (M = 17.00, SD = 2.00), and 135 μM (M = 7.00, SD = 1.00) was significantly different than the control (M = 100.00, SD = 0.00) at 48 h. **P < 0.01 vs control of 24h; ## P < 0.01 vs control of 48 h.
Western blotting. After being treated with PS VII (0.5, 1.5, and 4.5 μM) for 24 h, cells were washed twice with PBS and treated with extraction buffer (50 mM Tris– Cl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% deoxycholic acid). The cell extractions were collected and centrifuged at 10 000 ×g for 15 min at 4 °C, and the supernatants were collected as cell lysates. The cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were blocked with 5% (w/v) non-fat milk in PBS containing 0.05% Tween-20, and then blotted with primary antibody. Subsequently, the membranes were incubated with an appropriate secondary antibody (horseradish peroxidaseconjugated goat anti-mouse or anti-rabbit IgG). The immuno-detected proteins were then revealed by enhanced chemiluminescence. Statistics. All data were processed by SPSS 17.0. A oneway between subjects ANOVA was conducted to compare the effect of different doses of PSVII (0.5, 1.5, or 4.5 μM) on growth, migration, invasion, or expression of TIMP-1, 2 of A549 cells, followed by a post hoc multiple comparisons using Fisher’s least significant difference (LSD) t-test. Differences were considered significant at P < 0.05.
Figure 3. Effects of PS VII on cell migration. (A) Migration of cancer cell line A549 was assessed by wound-healing assay. Cells were cultured to nearly confluent cell monolayer. A scratch wound was created on the cell surface using a micropipette tip, and then PS VII (0.5, 1.5, and 4.5 μM) were added. The cultures were incubated at 37 °C and photographed with microscope at 0 h and 24 h. (B) The cell wound closure rate was measured at 24 h and was calculated according to the equation: Wound closure % = [1 (wound area at Tt / wound area at T0)] × 100%, where Tt is the time after wounding and T0 is the time immediately after wounding. The experiments were performed in triplicate. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions [F(3, 8) = 18.28, p = 0.001]; Post hoc comparisons using the LSD test indicated that the mean score for the 1.5 μM (M = 73.00, SD = 3.61) and 4.5 μM (M = 83.00, SD = 3.00) was significantly different than the control (M = 67.00, SD = 3.00). However, 0.5 μM (M = 66.00, SD = 3.00) did not significantly differ from control. **P < 0.01 vs control. Copyright © 2015 John Wiley & Sons, Ltd.
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RESULTS PS VII inhibited cell viability of A549 cells The viability of A549 cells treated with PS VII at different concentrations (0.5, 1.5, 5, 15, 45, and 135 μM) for 24 h and 48 h was determined by MTT assay. As illustrated in Fig. 2, PS VII decreased cell viability rate in a concentration-dependent manner. A higher concentration of PS VII would inhibit the growth of A549 cells directly, then, in order to observe the effect of PS VII on migration and invasion of A549 cells more accurately and clearly, the concentrations of 0.5, 1.5, and 4.5 μM (showed no obvious cytotoxicity to cells proliferation) were chosen in the following steps. PS VII inhibited the migration of cells in vitro One characteristic of tumor metastasis is the increased ability of tumor cells migration. The inhibition of A549 cell migration by PS VII was investigated by wound-healing assays. As presented in Fig. 3A, after incubation with different concentrations of PS VII for 24 h, higher concentration led to significantly inhibition of cell migration (P < 0.01). The results are shown in Fig. 3B. PS VII inhibited A549 cell spreading Cell morphology was analyzed by scanning electron microscopy. As shown in Fig. 4, A549 cells treated with solvent were spreading out with long filopodia and flat lamellipodia. In contrast, some cells became round with little membrane protrusions after PS VII (1.5 μM) treatment. The number of cell filopodia and lamellipodia reduced significantly. PS VII inhibited invasion of A549 cells in vitro In order to determine the inhibitory effect of PS VII on the invasion of A549 cells across the extracellular matrix (ECM), the cells that invaded through Matrigel-coated polycarbonate filter in the transwell chamber were analyzed. The results are shown in Fig. 5. Fig. 5A showed that the majority of A549 cells in control group invaded from the upper chamber to the lower chamber. However, the penetration of the
Figure 5. Effect of PS VII on invasion of A549 cells. (A) Cells were treated with various concentrations of PS VII (0.5, 1.5, and 4.5 μM) for 24 h, and cell invasion assay was performed. The invaded cells were photographed (100 × magnification). (B) The invaded cells were quantified by the absorbance of the crystal violet washed with 33% acetic acid from the cells that invaded the underside of the porous polycarbonate membrane. The experiments were performed in triplicate and represented the average of three experiments ± S.D. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions [F(3, 8) = 107.04, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.69, SD = 0.05), 1.5 μM (M = 0.51, SD = 0.02), and 4.5 μM (M = 0.36, SD = 0.03) was significantly different than the control (M = 0.97, SD = 0.07). **P < 0.01 vs control.
Matrigel coated filter by A549 cells was inhibited in the presence of PS VII. The quantification of cells in the lower chamber from Fig. 5B indicated that PS VII significantly inhibited A549 cells invasion (P < 0.01), and this inhibitory effect was concentration dependent.
Figure 4. Effects of PS VII on cell spreading. Cell morphology was analyzed by scanning electron microscopy (1.5k×). Copyright © 2015 John Wiley & Sons, Ltd.
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PS VII suppressed activity of MMP-2 and MMP-9 To investigate MMP-2 and -9 activities in A549 cells, the conditioned medium was subjected to gelatin zymography. The gelatinolytic activity at 72 and 92 kDa, which corresponded to the molecular mass of MMP-2 and -9, was detected in the conditioned medium from cells treated with PS VII. Fig. 6 illustrated that the activity of MMP-2 and -9 was markedly repressed by PS VII in a concentrationdependent manner.
PS VII affected metastasis-related protein levels in A549 cells Because the enzyme activity of MMP-2 and -9 was markedly repressed by PS VII, the effect of PS VII on the expression of MMPs was investigated by Western blot. After incubation with different concentrations of PS VII for 24 h, PS VII did not suppress the expression of MMP-2 and -9 (data not shown). So we further detected the expression of TIMP-1 and TIMP-2, which are two regulatory factors of the enzyme activity of MMPs. Fig. 7 indicated the levels of TIMP-1 and TIMP-2 were up-regulated by PS VII (P < 0.01). Similarly, Fig. 8 showed the protein concentration of
Figure 6. Effect of PS VII on activations of MMP-2 and -9 in A549 cells. (A) Cells were incubated for 24 h with a vehicle control or PS VII (0.5, 1.5, and 4.5 μM). Conditioning media were used for the measurement of MMP-2 and MMP-9 enzyme activity levels by gelatin zymography as described in Materials and methods. (B) Densitometry showing different activities of MMP-2 and MMP-9 in medium samples is presented in A. The experiments were performed in triplicate and represented the average of three experiments ± S.D. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions for MMP-2 [F(3, 8) = 87.94, p = 0.000], or for MMP-9 [F(3, 8) = 117.17, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 9.23, SD = 1.10), 1.5 μM (M = 5.43, SD = 0.38), and 4.5 μM (M = 2.37, SD = 0.31) was significantly different than the control (M = 12.00, SD = 1.00) for MMP-2; Post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 25.33, SD = 1.53), 1.5 μM (M = 19.33, SD = 1.53), and 4.5 μM (M = 10.33, SD = 1.53) was significantly different than the control (M = 31.00, SD = 1.00) for MMP-9. **P < 0.01 vs control. Copyright © 2015 John Wiley & Sons, Ltd.
Figure 7. Effect of PS VII on the expression of TIMP-1, TIMP-2, and E-cadherin. (A) Cells were treated with PS VII for 24 h, and the protein expression levels were estimated by Western blot. (B) Determined levels of TIMP-1, TIMP-2, and E-cadherin were quantified by densitometric analysis. The densitometric results are expressed as mean ± S.D. of three independent experiments. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions for TIMP-1 [F(3, 8) = 105.66, p = 0.000], for TIMP-2 [F(3, 8) = 261.64, p = 0.000], or for E-cadherin [F(3, 8) = 382.24, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.29, SD = 0.05), 1.5 μM (M = 0.35, SD = 0.04), and 4.5 μM (M = 0.68, SD = 0.05) was significantly different than the control (M = 0.14, SD = 0.02) for TIMP-1. However, did not significantly differ from control; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.33, SD = 0.04), 1.5 μM (M = 0.83, SD = 0.05), and 4.5 μM (M = 0.93, SD = 0.04) was significantly different than the control (M = 0.17, SD = 0.04) for TIMP-2; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 0.66, SD = 0.03), 1.5 μM (M = 1.56, SD = 0.06), and 4.5 μM (M = 1.96, SD = 0.12) was significantly different than the control (M = 0.25, SD = 0.04) for E-cadherin. **P < 0.01 vs control.
TIMP-1 and TIMP-2 in the conditioned media was also up-regulated by PS VII (P < 0.01). These results suggest that PS VII may affect the activity of MMPs by elevating the expression of TIMPs, which in turn led to the inhibition of invasion. E-cadherin is one of the main epithelial adhesive molecules. Loss of E-cadherin is associated with metastasis Phytother. Res. 29: 1366–1372 (2015)
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Figure 8. Supernatant from cultured A549 cells was collected and used to determine secreted TIMP1/2 protein levels after the cells were treated with PS VII for 24 h by using a human TIMP1/2 enzyme-linked immunosorbent assay (ELISA). TIMP1/2 concentrations (ng/mL) were adjusted to the number of cultured cells. Error bars represent the S.D. from five independent experiments. There was a significant effect of different doses of PS VII at the p < 0.05 level for the four conditions for TIMP-1 [F(3, 8) = 40.89, p = 0.000], for TIMP-2 [F(3, 8) = 107.23, p = 0.000]; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 41.67, SD = 1.53), 1.5 μM (M = 46.33, SD = 1.53), and 4.5 μM (M = 51.33, SD = 2.52) was significantly different than the control (M = 34.33, SD = 2.08) for TIMP-1; post hoc comparisons using the LSD test indicated that the mean score for the 0.5 μM (M = 59.33, SD = 1.53), 1.5 μM (M = 66.67, SD = 1.53), and 4.5 μM (M = 77.67, SD = 1.53) was significantly different than the control (M = 55.00, SD = 2.00) for TIMP-2. **P < 0.01 vs control.
and poor prognosis in many neoplasms (Buda and Pignatelli, 2011). We found that PS VII treatment could up-regulate E-cadherin levels effectively.
DISCUSSION Suppression of cancer metastasis is an urgent therapeutic need in treatment of lung cancer. But most existing drugs only inhibit cancer cell proliferation. Little has been accomplished in terms of treating cancer metastasis. In our previous study, we have found that PS VII, a saponin compound isolated from T. tschonoskii Maxim., has growth inhibitory effect in vitro as well as in vivo (Li et al., 2014). In addition, non-apoptotic processes, including inhibition of cancer metastasis (Yoon et al., 2012), were also involved in saponin cytotoxic activity. In the current study, we therefore investigated the anti-metastasis activity of PS VII using lung cancer A549 cells. Metastasis is a complex, multistep process. It includes migration, invasion, adhesion, infiltration, colonization at a distant site, and the subsequent formation of new microvessels (Weng and Yen, 2012). To successfully invade, cells must acquire the ability of migration. In a non-cytotoxic dose, we evaluated the anti-migratory activity of PS VII through the wound healing assay which was a classic in vitro assay of cell migration. Our results showed that PS VII could reduce cell migration rate in a concentration-dependent manner. Cell migration is a highly integrated multistep process, which is initiated by the formation of filopodia and lamellipodia in the cell membrane (Bailly and Condeelis, 2002). Copyright © 2015 John Wiley & Sons, Ltd.
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Migration of cells is based on cycles of lamellipodial extension, attachment, cell body translocation, and retraction of the cell (Berrier and Yamada, 2007). We found PS VII-treated A549 cells became round, with little membrane protrusions. The number of cell filopodia and lamellipodia reduced significantly, suggesting the motility of cancer cells were inhibited. Cell migration partly depends on efficiency of cell attachment and detachment between cells (Mitra et al., 2005). E-cadherin is an essential component of adherence junctions. E-cadherin binds with its extracellular domain to an E-cadherin molecule of the neighboring epithelial cell to stabilize cell-to-cell contacts. So we detected the expression of E-cadherin and the results showed that E-cadherin was up-regulated after the cells were treated with PS VII. This may be one of the reasons for the reduced cell migration rate. In this study, the invasive ability of cells was evaluated by transwell invasion assay. The results demonstrated that PS VII could inhibit cell invasion rate in a concentration-dependent manner. Cell invasion requires proteolysis of extracellular matrix (ECM) components. In these steps, expression of proteolytic enzymes, such as MMPs, is crucial for ECM degradation (Friedl and Wolf, 2003). Many studies have demonstrated that higher protein expression levels of MMP-2 and -9 were associated with a poorer prognosis (Lin et al., 2011). Therefore, we investigated whether the inhibitory effect of PS VII on cell invasion was through suppressing expression of MMP-2 and -9. Results from gelatin zymography showed that PS VII could suppress the proteolytic enzyme activity of MMP-2 and -9. However, the expression of these two proteins was not affected by PS VII. Activities of MMPs are controlled by their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs) such as TIMP-1 and TIMP-2 (Di Carlo, 2014), so we further detected the expression of TIMP-1 and -2. We found that TIMP-1 and -2 were up-regulated by PS VII treatment, suggesting that PS VII may affect the activity of MMPs by elevating the expression of TIMPs. Taken together, our data suggested that PS VII obtained from T. tschonoskii Maxim. could inhibit migration and invasion of A549 cells. The increase in the expression of TIMP-1/2 and E-cadherin may contribute to this effect. Base on our study, PS VII could be a promising agent for therapeutic and preventive purposes of lung cancer by not only suppressing the proliferation of cells but also inhibiting metastasis-associated events.
Acknowledgements We thank the Department of forestry of Shaanxi Province and the Taibaishan natural preserve of Shaanxi Province for helping us collect the Trillium tschonoskii Maxim. This investigation was supported by the grant from the Taibaishan Natural Preserve of Shaanxi Province, and the grant (No. 81302787) from the National Nature Science Foundation of China, and the grant (Nos. 2012M512102, 2013T60964) from the Postdoctoral Science Foundation of China.
Conflict of Interest The authors have declared that there is no conflict of interest. Phytother. Res. 29: 1366–1372 (2015)
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Phytother. Res. 29: 1366–1372 (2015)
