Suppression of the migration and invasion is mediated by triptolide in B16F10 mouse melanoma cells through the NF-kappaB-dependent pathway Hui-Yu Jao1, Fu-Shun Yu2, Chun-Shu Yu3, Shu-Jen Chang3, Kuo-Ching Liu4, Ching-Lung Liao5, Da-Tian Bau6,7,*, and Jing-Gung Chung1,8,* 1 Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan; 2 Department of Dentist, China Medical University, Taichung 404, Taiwan; 3 School of Pharmacology, China Medical University, Taichung 404, Taiwan; 4 Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan, ROC 5 Graduate Institute of Chinese Medicine, China Medical University, Taichung 404, Taiwan, ROC 6 Institute of Clinical Medical Science, China Medical University, Taichung 404, Taiwan; 7 Terry Fox Cancer Research laboratory, China Medical University Hospital, Taichung 404, Taiwan; 8 Department of Biotechnology, Asia University, Taichung 413, Taiwan. Running title: Triptolide inhibited migration and invasion of B10F6 melanoma cells * These authors contributed equally to this work. Competing Interests: The authors have declared that no competing interests exist. *Address correspondence to Prof. Jing-Gung Chung, Department of Biological Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: +886-4-2205 3366 ext 2531, fax: +886-4-2205 3764. E-mail address: [email protected] *Address correspondence to Prof. Da-Tian Bau, Terry Fox Cancer Research Lab., China Medical University Hospital, 2 Yuh-Der Road, Taichung 404, Taiwan. Tel: +886 422052121 ext 7534. Email address: [email protected] Abstract. Melanoma cancer is one of the major causes of death in humans worldwide. Triptolide is one of the active components of Tripterygium wilfordii Hook F, and has biological activities including induced cell cycle arrest and induction of apoptosis but its antimetastatic effects on murine melanoma cells have not yet been elucidated. Herein, we investigated the effect of triptolide on the inhibition of migration and invasion and possible associated signal pathways in B16F10 murine melanoma cancer cells. Wound healing assay and Matrigel Cell Migration Assay and Invasion System demonstrated that triptolide marked inhibiting the migration and invasion of B16F10 cells. Gelatin zymography assay demonstrated that triptolide significantly inhibited the activities of matrix metalloproteinases-2 (MMP-2). Western blotting showed that triptolide markedly reduced CXCR4, SOS1, GRB 2, p-ERK, FAK, p-AKT, Rho A, p-JNK, NF-B, MMP-9 and MMP-2 but increased PI3K and p-p38 and COX2 after compared to the untreated (control) cells. Real time PCR indicated that triptolide inhibited the gene expression of MMP-2, FAK, ROCK-1 and NF-B but did not significantly affect TIMP-1 and -2 gene expression in B16F10 cells in vitro. EMSA assay also showed that triptolide inhibited NF-B DNA binding in a dose-dependent manner. Confocal laser microscopy examination also confirmed that triptolide inhibited the expression of NF-B in B16F10 cells. Taken together, we suggest that triptolide inhibited B16F10 cell migration and invasion via the inhibition of NF-B expression then led to suppress MMP-2 and -9 expressions. Keywords: migration; invasion; triptolide; B16F10 mouse melanoma cells; NF-kappaB-dependent pathway. Introduction Malignant melanoma has been recognized to be one of the most lethal and aggressive malignancies in human (Siegel et al., , 2013) and the incidence of melanoma are worldwide. Metastatic melanoma is characterized by a high mortality rate (Garbe and Leiter, 2009). Patients with malignant melanoma were hard to treat due to resistance to standard chemotherapy (Jilaveanu et al., , 2009). Currently, the treatments of melanoma in clinical patients are surgery, chemotherapy, and radiotherapy or a combination of these therapies. However, the cure rate is still unsatisfactory. Thus, there is an urgent need to find new compound or novel treatments for melanoma patients. After compared to basal cell carcinoma and squamous cell carcinoma, melanoma has a higher metastatic potential. Metastasis is one of the factors for affecting the treatment of cancer and is associated with mortality with metastatic diseases rather than the primary tumor (Tang et al., , 2012). Metastasis is a multistep process, at first cancer cells to detach from primary tumor, then to migrate, adhesive and invade to the blood or lymphatic vessels and they need matrix metalloproteinases (MMPs) for cancer cells to the target tissues or organs. Thus, MMPs play important roles for metastasis formation. It is well documented that the expression and activity of MMPs can enhance many types of human cancers with advanced tumor stage and shortened survival (Egeblad and Werb, 2002). Therefore, inhibition of MMPs of cancer cells may lead to improve cancer therapy in patients (Egeblad and Werb, 2002). Triptolide, a diterpene triepoxide, is one of the components from the Chinese herb Tripterygium wilfordii, and has been shown to induce cell death in many human cancer cells such as breast cancer, lung cancer, pancreatic cancer, bladder cancer, and cervical cancer and colorectal carcinomas (Banerjee et al., , 2013; Chen et al., , 2014; Clawson et al., , 2010; Jiang et al., , 2001; Kim et al., , 2010; Li et al., , 2010; Liu et al., , 2009; Yang et al., , 2003) including melanoma cancer cells (Hung et al., , 2013). Triptolide have been shown to inhibit human melanoma A375 cells proliferation and induce apoptosis through a NF-B-mediated mechanism (Tao et al., , 2012). It was reported that triptolide reduced pancreatic cell growth and metastases of tumors in vivo (Mujumdar et al., , 2010). Triptolide have been shown to inhibit experimental metastasis of B16F10 cells to the lungs and spleens of mice (Yang et al. , 2003). Triptolide induces down-regulation of hST8Sia I gene expression through NF-κB activation in human melanoma cells (Kwon et al., , 2010). Although triptolide have been shown to induce cell death of human melanoma and mouse melanoma cells, however, there is no available information to show triptolide inhibits migration and invasion of mouse melanoma cell. Thus, we investigate the effects of triptolide on migration and invasion of mouse melanoma B16F10 cells in vitro. The present results clearly indicate that triptolide inhibited the migration and invasion of B16F10 melanoma cells through the inhibition of the NF-B signal pathways. Materials and Methods Chemicals and reagents. Triptolide, dimethyl sulfoxide (DMSO), propidium iodide (PI), Tris-HCl, Trypsin, Trypan blue and -Actin were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). Control cultures received the carrier solvent (0.5% DMSO). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin was obtained from Invitrogen (Carlsbad, California, USA). Triptolide was dissolved in DMSO and stored at -20˚C. Antibodies against CXCR4 was obtained from Novus (Novus Biologicals, Littleton, CO, USA). MMP-9, MMP-2, p-ERK, p-JNK, p-p38, PKC, p-AKT, NF-B and Rho A were purchased from Santa cruz (Santa Cruz Biotechnology, Inc. Calif, USA). SOS1, GRB2, Ras, PI3K, COX2 were obtained from BD (BD Biosciences, Heidelberg, Germany). FAK was obtained from Upstate (Upstate, Lake Placid, NY). Cell culture and viability assay. B16F10 mouse melanoma cancer cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in DMEM medium supplemented with 10% FBS, 0.1 mg/mL streptomycin, and 100 units/mL penicillin at 37˚C under 5% CO2 for 24 h. Cells (2x105 cells/well) were plated in 12-well plates with DMEM medium for 24 h and then were treated with 0, 5, 10, 15, 20, 25, 30 and 35 nM of triptolide and were incubated at 37°C, 5% CO2 and 95% air for 24 and 48 h. After incubation, cells were examined under contrast phase microscopy and were collected for viability determination, and isolated cells were stained with PI (5 μg/ml) and analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA) as described previously (Kuo et al., , 2014; Wu et al., , 2011; Yeh et al., , 2013). 4 Adhesion assay. B16F10 cells (5×10 cells/well) were placed in 96-well plates pre-coated with type I collagen (10 μg/mL) (Millipore, Temecula, CA, USA) for 60 min at 37°C and then were incubated with triptolide (0, 5, 10 and 20 nM) for 24 or 48 h at 37°C. After incubation, unattached cells were removed, and attached cells were mixed in 1% glutaraldehydein in PBS for 20 minutes, and stained with 0.02% crystal violet solution for 5 minutes at room temperature. And then 70% ethanol was used to dissolve crystal violet, and O.D. was measured at 570 nm by using microplate reader as described previously (23). Total percentage of adhesion was calculated based on the adhesion cells compared to the control (Lu et al., , 2012). 5 Wound healing assay. B16F10 (3×10 cells/well) cells were placed on 6-well plates for 24 h and then were wounded with scratched with a 200 l pipette tip as previously described (Lu et al. , 2012). After wounding, medium was changed and 0, 5, 10 and 20 nM of triptolide was added to the medium. After incubation for 0, 12, 24 and 36 h, wounding areas were examined and photographed under contrast phase light microscopy at x200. Migrated cells that had moved to the wounded areas were counted under a microscope for quantification of cell migration (Lu et al. , 2012). Cell migration and invasion assay. Cell invasion was measured by Matrigel (BD Biosciences, Franklin Lakes, New Jersey, USA) -coated transwell cell culture chambers (8 mm pore size; Millipore, Billerica, Massachusetts, USA). B16F10 cells was kept for 24 h in serum-free DMEM medium and were collected and placed in the upper chamber of the transwell insert (5 x104 cells/well), and triptolide was added to the well at the final concentration 0, 5, 10 and 20 nM and DMEM containing 10% FBS was added to the lower chamber. The plates were incubated at 37°C in a humidified atmosphere with 95% air and 5% CO2 for 24 or 48 h, and non-invasive cells in the upper chamber were removed and invasive cells in the bottom were fixed with 4% formaldehyde in PBS and stained with 2% crystal violet in ethanol and then were counted under a light microscope at x200. For cell migration measurements that were done as cell invasion assay, everything was the same except for no Matrigel-coating of filter membrane. Cells located on the underside of the filter were photographed and counted under a light microscope at x200 (Lu et al. , 2012). Gelatin zymography assay. The conditioned medium of triptolide-treated B16F10 cells (1x107 cells/ml) at the final concentrations of 0, 5, 10 and 20 nM for 24 and 48 h was collected and concentrated using ultrafiltration spin columns fitted with 30 kDa MWCO membranes (Sartorius Stedim Biotech GmbH, Goettingen, Germany). The harvest medium was separated by dilution in zymographic sample buffer and was analyzed on 7.5% polyacrylamide gels containing 0.1% gelatin (Sigma-Aldrich). The gel was then incubated with development buffer (50 mM Tris, pH 7.5; 200 mM NaCl; 5 mM CaCl2, 1 µM ZnCl2 and 0.02% Brij-35) at 37°C for 18 h. Gels were stained with 0.2% Coomassie blue G-250 for 3 h, and areas of gelatinolytic activity were visualized as transparent bands and analyzed by NIH image software as described previously (Lu et al. , 2012). Western blotting analysis. B16F10 cells (1x106 cells/well) were placed in 6-well plates for 24 h and were incubated with triptolide (0, 5, 10 and 20 nM) for 24 and 48 h. Cells were harvested and were lysed in ice-cold 50 mM potassium phosphate buffer (pH 7.4) containing 2 mM EDTA and 0.1% Triton X-100 for sonication. The homogenate was centrifuged and supernatant was collected and quantitated using Bio-Rad protein assay kit (Hercules, California, USA) with bovine serum albumin (BSA) as the standard as described previously (Kuo et al. , 2014; Lu et al. , 2012). A total of 20 μg of protein was separated using 10%-15% SDS-PAGE and then electrophoretically transferred to a PVDF membrane (Millipore, Temecula, CA, USA), blotted with the relevant antibodies such as anti-CXCR4, SOS1, GRB 2, Ras, p-ERK, FAK, PKC, PI3K, p-AKT, Rho A, p-JNK, p-p38, NF-B, COX2, MMP-2, MMP-9. After washing three times with TBST, the membrane was incubated with horseradish peroxidase–conjugated secondary antibody diluted in TBST. These blots were detected by an enhanced chemiluminescence reagent (ECLTM, Amersham Biosciences, Piscataway, NJ, USA). The relative protein bands obtained were quantified using NIH Image analyzer (NIH, Bethesda, MD, USA) (Kuo et al. , 2014; Lu et al. , 2012). 6 Real-time PCR of metastasis-related mRNA expressions. B16F10 cells (1×10 cells/well) were placed in 6-well plates for 24 h and then were incubated with triptolide (0 and 20 nM) for 24 h. Cells were harvested and total RNA was extracted and RNA samples were performed for cDNA reverse transcription according to the protocol of the supplier (Applied Biosystems, Foster City, CA, USA). The primers were MMP-2-F: CCCCAGACAGGTGATCTTGAC; GCTTGCGAGGGAAGAAGTTG; FAK-R: FAK-F: MMP-2-R: TGAATGGAACCTCGCAGTCA; TCCGCATGCCTTGCTTTT; Rock-1-F: ATGAGTTTATTCCTACACTCTACCACTTTC; Rock-1-R: TAACATGGCATCTTCGACACTCTAG; NF-B-F: AGTTGAGGGGACTTTCCCAGGC; NF-B-R: TCAACTCCCCTGAAAGGGTCCG; TIMP-1-F: TGTTTATCCATCCCCTGCAAA; TIMP-R: CAAGGTGACGGGACTGGAA; TIMP-2-F: GGGCCAAAGCGGTCAGT; TIMP-2-R: TTGAACATCTTTATCTGCTTGATCTCA; GAPDH-F: ACACCCACTCCTCCACCTTT; GAPDH-R: TAGCCAAATTCGTTGTCATACC. Each assay was performed in triplicate by the Applied Biosystems 7300 Real-Time PCR system, and the expression (density of each band) fold changes were performed by comparative CT (threshold cycle) method (Liu et al., , 2013). Electrophoretic mobility shift assay (EMSA). B16F10 cells (1x107 cells/dish) were treated with triptolide (20 nM) for 0, 6, 9 and 12 h or were treated with 0, 5, 10 and 20 nM of triptolide for 12 h. Nuclear extracts from each treatment and time periods were prepared by using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce) as described previously (Kuo et al., , 2009). Each protein concentration was quantitated and biotin end-labeled oligonucleotide sequences were 5′-Biotin-GATCCAGGGGACTTTCCCTAGC-3′ corresponding to the consensus site of NF-κB. Nuclear extract proteins (5 μg) from all samples were used for EMSA with LightShift Chemiluminescent EMSA Kit according to the manufacturer’s protocol. The nuclear extracts or purified factor were incubated with biotin end-labeled duplex DNA and electrophoresed on a 6% polyacrylamide native gel and transferred onto nylon membrane and detection was performed following the manual of the ECL kit (Kuo et al. , 2009). Immuno-staining of NF-κB and was examined by confocal laser scanning microscopy. B16F10 cells (5 x 104 cells/well) were placed in four-well chamber slides and treated with triptolide (0 and 20 nM) for 9 h and then were ﬁxed in 3% formaldehyde in PBS for 15 min, followed by using 0.1% Triton X-100 in PBS for 1 h to permeable cells and 2% BSA was used to block the nonspeciﬁc binding sites. Cells were incubated with primary antibody anti-NF-κB (1:200 dilution) and were stained with secondary antibody FITC-conjugated goat antimouse IgG (1:100 dilution) (green ﬂuorescence), followed by using PI (red ﬂuorescence) counterstaining nuclei. Cells were examined and photomicrographed using a Leica TCS SP2 confocal spectral microscope as described previously (Kwon et al. , 2010). Statistical analysis. Data are presented as mean ± standard deviation. Student's t-test was used for comparison of quantitative real-time PCR data and other data. Differences between the triptolide-treated and untreated control groups were compared by Student’s t test. A p value of less than 0.05 was considered statistically significant. Results Triptolide decreased the percentage of viable B16F10 mouse melanoma cancer cells. We evaluated the effects of triptolide (0, 5, 10, 15, 20, 25, 30 and 35 nM) in B16F10 cells at 24 and 48 h post-treatment. The results are shown in Figure 1. Melanoma cells showed growth inhibition and reduced cellular viability in a dose-dependent manner. Increased time of treatment lead to decreased cell viability. Triptolide inhibited the adhesion of B16F10 cells in vitro. B16F10 cells were treated with triptolide at 0, 5, 10 and 20 nM for 24 and 48 h and were measured for the inhibition of cell adhension by using ELSIA reader and the results are shown in Figure 2. The results of the assay showed that triptolide significantly inhibited the adhesion of B16F10 cancer cells studied. Cell adhension was blocked by as low concentration of triptolide at 10 nM for 48 h treatment. However, triptolide at 20 nM decreased cell adhension at 24 and 48 h treatment. Triptolide suppressed the migration of B16F10 cells in vitro. Wound-healing assay was used to investigate the migration of B16F10 cells cultured with or without triptolide (0, 5, 10 and 20 nM) for 12, 24 and 36 h. Results shown in Figure 3 indicated that triptolide significantly reduced the migration of B16F10 cells in a doseand time-dependent manner during 6-12 h. Triptolide inhibited the migration and invasion of B16F10 cells in vitro. In order to further investigate the cell migration and invasion and whether or not they were affected by triptolide, B16F10 cells were placed on Millicell chambers with uncoated (for migration) or matrigel-coated (for invasion) filters and then triptolide (0, 5, 10 and 20 nM) was added to the cells and then were incubated for 24 and 48 h. Cell migration and invasion were examined, counted and photographed as shown in Figure 4A, B, C and D. Results demonstrated that triptolide significantly inhibited the migration and invasion of B16F10 cells and these effects are dose-dependent that was in agreement with results from wound healing assay (Figure 3). Triptolide inhibited the matrix metalloproteinases-2 activities of B16F10 cells. For investigating triptolide suppressed cell mobility (migration and invasion) and whether or not they involved the reduction of MMP-2 activities, they were measured by using gelatin zymography assay and the results shown in Figure 5. Results indicated that triptolide inhibited the MMP-2 activities at 5, 10 and 20 nM for 48 h, and these effects are in a dose-dependent manner. Triptolide affect the protein levels of associated with migration and invasion of B16F10 cells. We investigated triptolide suppressing migration and invasion of B16F10 cells and whether they mediated the altered levels of associated protein with migration and invasion. After Western blotting examination, the representative figures were showed in Figure 6A, B and C. The results indicated that triptolide markedly reduced CXCR4, SOS1, GRB 2, p-ERK (Fig. 6A); FAK and p-AKT (Fig. 6B); Rho A, p-JNK, NF-B, MMP-9 and MMP-2 (Fig. 6C) but increased PI3K (Fig. 6B) and p-p38 and COX2 (Fig. 6C) after compared to the untreated (control) cells. Triptolide affect gene expression of B16F10 cells. B16F10 cells were treated with triptolide (0 and 20 nM) for 24 h. Cells were harvested and total RNA was extracted and RNA samples were performed for cDNA reverse transcription and the results are shown in Figure 7. Results indicated that triptolide inhibited the gene expression of MMP-2, FAK, ROCK-1 and NF-B but did not significantly affect TIMP-1 and -2 gene expression in B16F10 cells in vitro. Triptolide inhibits the NF-κB signaling pathways in B16F10 cells. The effect of triptolide on DNA binding of NF-κB was determined using EMSA. B16F10 cells as they are after exposure to 20 nM of triptolide for 6, 9 and 12 h or exposed to triptolide (0, 5, 10 and 20 nM) for 12 h. The results are shown in Fig. 8A and B, which demonstrated that triptolide inhibited NF-κB DNA binding in a dose-dependent manner. Binding of NF-κB was particularly inhibited by treatment with 20 nM of triptolide. Therefore, we suggest that triptolide might block the invasion, migration and adhesion in B16F10 cells by suppressing the NF-κB signaling. Triptolide inhibits the NF-κB protein levels in B16F10 cells. For further confirming triptolide inhibits NF-B levels in B16F10 cells. The protein expressions of NF-κB in B16F10 cells was examined by confocal laser system microscopy and the results are shown in Figure 9. The results indicated that triptolide markedly inhibited the NF-κB protein levels in cytosol but did increase the protein levels in nuclei. Discussion Triptolide is known to induce cell death through cell cycle arrest and induction of apoptosis in numerous malignancies cells (Corson and Crews, 2007). Although it was reported that triptolide inhibited human colon rectal cancer cell migration as well as proliferation (Johnson et al., , 2011) and inhibited migration and invasion of ovarian cancer cells (Zhao et al., , 2012), however, there is no available information to show triptolide affecting cell mobility such as migration and invasion of mouse melanoma B16F10 cells. Thus, we went on to identify the mechanistic background of this phenomenon and, we further examined the migration and invasion of B16F10 cells in vitro after exposed to various concentrations of triptolide and the results indicated that the adhension, migration and invasion were inhibited (Figs. 2 and 3). In this report we show for the first time that triptolide inhibits the adhesion, migration and invasion of melanoma cancer B16F10 cell, hereby suppressing the very initial stage of cancer cell migration. These finding are in agreement with other reports shown that triptolide inhibited ovarian cancer cell migration and invasion in vitro. Matrix metalloproteinases (MMPs) have been recognized to play an important role in cell motility and invasion via to degrade extracellular matrix (ECM) components. Degradation of ECM is a critical step in cancer cell invasion into neighboring tissues or organs and also for cancer cell to initiate metastasis. In the pathogenesis of diverse diseases, such as periodontitis, rheumatoid arthritis, and skin wound healing, it has been shown that the MMPs were induced or over-expressed (Kanbe et al., , 2011; Sorsa et al., , 2004; Vu and Werb, 2000). It was reported that triptolide significantly inhibited the migration and invasion of ovarian cancer SKOV3 and A2780 cells by suppression of MMP7 and MMP19 and up-regulation of E-cadherin expression (Johnson et al. , 2011). To this end we examined the effect of triptolide on the expression of associated proteins involved in B16F10 melanoma cancer cell adhesion, migration and invasion such as MMP-2, MMP-9 and down-regulated major factor of NF-B. Our results showed that triptolide inhibited the protein expression of MMP-2 and -9 (Fig. 6C). MMP-2 and MMP-9 have the ability to degrade collagen IV and V and counteract fibrosis (Sato et al., , 1994). It was reported that MMP activity is regulated via tissue inhibitors of metalloproteinases (TIMPs) which form complexes with MMPs for inhibiting the active form of enzymes (Mazanowska et al., , 2014). Results showed that triptolide did not significantly affect the expression of TIMP-1 and TIMP-2 (Fig. 7). Rho A is a critical factor and also plays an important roles for cells cytoskeleton reorganization, adhesion and motility (Jackson et al., , 2011). Results from Figure 6C have shown that triptolide significantly decreased the expressions of Rho A and p-JNK in B16F10 cells. Furthermore, triptolide also inhibited the protein expressions of FAK and p-AKT (Fig. 6B) and p-ERK (Fig. 6A). Thus, we suggest that triptolide-induced MMP-9 expression is primarily regulated by PKC, PI3K/Akt, p-ERK, and p38 MAPK. CXCR4 expression exists in many types of human cancers and it is generally recognized and associated with metastasis (Ruffini et al., , 2007). It was reported to play an essential role of SOS1/EPS8/ABI1 tri-complex in cell migration and metastasis (Chen et al., , 2010). It was also reported that GRB2 expression status could be as a positive biomarker of esophageal squamous cell carcinoma (ESCC) progression and lymph node metastasis (Li et al., , 2014). Furthermore, it was demonstrated that the blockage of the Ras-ERK pathway will lead to decreased cell migration and invasion (Li et al. , 2014). In the present study, we showed that triptolide inhibited the protein levels of CXRC4, SOS1, GRB2 but did not significant affect Ras (Fig. 6A). The results from figure 6C showed that triptolide down-regulated NF-B protein levels in B16F10 cells. Furthermore, we also used confocal laser microscopy examination to confirm triptolide inhibited the NF-B expression (Fig. 9). In order to investigate triptolide inhibited NF-B protein expression, we used real time PCR which indicated that triptolide inhibited the gene expression of NF-B (Fig. 7). We also used EMSA method to show that triptolide inhibited the NF-B-DNA binding (Fig. 8). Thus, we suggest that triptolide inhibited NF-B to binding DNA promoter then inhibited MMP-9 expression in B16F10 cells. It was reported that MMP-9 promoter contains cis-acting regulatory elements for transcription factors NF-B site (Huang et al., , 2014). In summary, we found that triptolide significantly inhibited migration and invasion in murine melanoma B16F10 in vitro with by suppression of MMP-2 and MMP-9 expressions. We also found that triptolide inhibited Rho A, SOS1/GRB2, AKT and FAK to lead to suppress migration and invasion of B16F10. Furthermore, triptolide down-regulated the NF-B expression through inhibited binding to promoter of NF-B and inhibited genes expression. These observations can be summarized in Figure 10. This finding suggests that triptolide is a potential candidate for the development of chemotherapeutic treatments for melanoma in the future. Declaration of Conflicting Interests The authors have no conflicts of interests. Acknowledgements This study is supported in part by a research grant from China Medical University [CMU102-ASIA-20]. Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China medical University, Taichung, Taiwan, R.O.C. References Banerjee S, Sangwan V, McGinn O, Chugh R, Dudeja V, Vickers SM, Saluja AK. 2013. Triptolide-induced cell death in pancreatic cancer is mediated by O-GlcNAc modification of transcription factor Sp1. J Biol Chem 288:33927-33938. Chen H, Wu X, Pan ZK, Huang S. 2010. Integrity of SOS1/EPS8/ABI1 tri-complex determines ovarian cancer metastasis. Cancer Res 70:9979-9990. 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Triptolide inhibited the cell-matrix adhension of B16F10 cells. 4 B16F10 cells (5×10 cells/well) were placed in 96-well plates pre-coated with type I collagen (10 μg/mL) (EMD Millipore) for 60 min at 37∘C and then were incubated with triptolide (0, 5, 10 and 20 nM) for 24 or 48 h at 37∘C. Unattached cells were removed, and attached cells were mixed in 1% glutaraldehydein in PBS for 20 minutes, and stained with 0.02% crystal violet solution for 5 minutes. And then 70% ethanol was used to dissolve crystal violet, and O.D. was measured at 570 nm by using microplate reader as described in Materials and Methods. *p < 0.05, significant difference between triptolide-treated and control groups as analyzed by Student’s t test. Figure 3. Triptolide inhibits the migration of B16F10 cell. Cells were kept on the 6-well dish for 24 h before a wound was created by a yellow pipette tip to scrape the confluent cell layers. Triptolide at various concentrations were added to the well then incubation for 24 h. A: Some of the representative photographs of invading treated and untreated cells are presented. B: the percentage of inhibition of cell migration was quantitated. *p < 0.05, significant difference between triptolide-treated and control groups as analyzed by Student’s t test. Figure 4. Triptolide suppressed the migration and invasion of B16F10 cells in vitro. Cells (5 x104 cells/well) were treated with 0, 5, 10 and 20 nM of triptolide for 24 h after cells penetrated through to the lower surface of the filter (without Matrigel) for migration measurement (A and B) or the filter (with Matrigel) for invasion measurements as described in Materials and Methods. Representative columns (mean) from three independent experiments. *p < 0.05, significant difference between triptolide-treated groups and the control as analyzed by Student’s t test. Figure 5. Triptolide affects the activities of matrix metalloproteinase-2 (MMP-2) activities in B16F10 cells. Cells were treated with 0, 5, 10 and 20 nM of triptolide and then were harvested and perform by gelatin zymography. A: representative zymogram from three independently experiment was used to detect the activity of secreted MMP-2. B: the different activity of MMP-2 was determined by densitometric analysis and results are expressed as a percentage of the control (100%). *p < 0.05, significant difference between triptolide-treated groups and the control as analyzed by Student’s t test. Figure 6. Triptolide affect the levels of associated proteins in migration and invasion of B16F10 cells. Cells (5 x105 cells/well) were treated with 0, 5, 10 and 20 nM of triptolide for 24 and 48 h and then cells were collected and the total protein extracted and determined as described in Materials and Methods section. A: CXCR4, SOS1, GRB2, p-ERK; B: Fak, PKC, PI3K, p-AKT; C: Rho A, p-JNK, NF-kB, MMP-9 and MMP-2, p-p38 and COX2 expressions were estimated by Western blotting as described in Materials and Methods. Figure 7. Triptolide affect gene expression of metastatic association in B16F10 cells. 6 Cells (1×10 cells/well) were placed in 6-well plates for 24 h and then were incubated with triptolide (0 and 20 nM) for 24 h. Cells were harvested and total RNA was extracted and RNA samples were performed for cDNA reverse transcription according to the protocol of the supplier as described in Materials and Methods. Each assay was performed in triplicate by the Applied Biosystems 7300 Real-Time PCR system. Figure 8. Triptolide inhibits the NF-κB signaling pathways in B16F10 cells. B16F10 cells after exposure to triptolide (0, 5, 10 and 20 nM) for 12 h or exposed to 20 nM of triptolide for 6, 9 and 12 h. Then cells were harvested for examining the DNA promoter binding assay by using EMSA as described in Materials and Methods. A: dose-dependent assay. B: Time-dependent assay. Figure 9. Triptolide affects the NF-κB p65 expression in B16F10 cells. Cells (5 x104 cells/well) were placed on 6-well chamber slides then were treated with 0 and 20 nM of triptolide for 9 h, then were fixed and stained using anti-NF-κB p65 then stained with a secondary antibody (green fluorescence) followed by nuclear counterstaining with PI (red fluorescence). Photomicrographs were obtained using a Leica TCS SP2 confocal spectral microscope as described in Materials and Methods. Figure 10. The possible signaling pathways for triptolide inhibited cell migration and invasion in B16F10 murine melanoma cells.