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Carcinogenesis vol.34 no.7 pp.1601–1610, 2013doi:10.1093/carcin/bgt065Advance Access publication February 20, 2013

Diallyl trisulfide inhibits proliferation, invasion and angiogenesis of osteosarcoma cells by switching on suppressor microRNAs and inactivating of Notch-1 signaling

Yonggang Li, Jingru Zhang1, Lei Zhang, Meng Si, Han Yin and Jianmin Li*

Department of Orthopedics and 1Department of Hematology, Qilu Hospital, Shandong University, Jinan 250012, China

*To whom correspondence should be addressed. Tel: +86 531 82169882; Fax: +86 531 86115887; Email: gklijianmin@gmail.com

Notch signaling pathway plays critical roles in human cancers, including osteosarcoma, suggesting that the discovery of specific agents targeting Notch would be extremely valuable for osteo-sarcoma. Our previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of osteosarcoma cells by triggering cell cycle arrest and apoptosis in vitro. However, the underlying mechanism is still unclear. In this study, we found that DATS suppressed cell survival, wound-healing capacity, invasion and angiogenesis in osteosarcoma cells. These effects were associated with decreased expression of Notch-1 and its downstream genes, such as vascular endothelial growth factor and matrix metallo-proteinases, as well as increased expression of a panel of tumor-suppressive microRNAs (miRNAs), including miR-34a, miR-143, miR-145 and miR-200b/c that are typically lost in osteosarcoma. We also found that reexpression of miR-34a and miR-200b by transfection led to reduced expression of Notch-1, resulting in the inhibition of osteosarcoma cell proliferation, invasion and angiogenesis. These results clearly suggest that DATS inhibited osteosarcoma growth and aggressiveness via a novel mechanism targeting a Notch–miRNA regulatory circuit. Our data provide the first evidence that the downregulation of Notch-1 and reex-pression of miRNAs by DATS may be an effective approach for the treatment of osteosarcoma.

Introduction

Osteosarcoma is a highly malignant bone cancer associated with locally aggressive growth and early metastatic potential. Intensive chemotherapy combined with aggressive surgical techniques have improved survival; however, patients with metastatic disease or with recurrent disease at time of diagnosis have an extremely poor prog-nosis, with only 20% surviving at 5 years (1,2). This disappointing outcome strongly suggests that the evaluation of novel, targeted thera-peutic agents is urgently needed to prevent osteosarcoma progression and improve patient survival rates. Identifying molecular signaling mechanisms involved in osteosarcoma carcinogenesis and metastasis may be the key to providing such novel treatment approaches.

Notch signaling pathway plays a pivotal role in fundamental pro-cesses of cell fate determination, including stem cell maintenance, differentiation, proliferation and apoptosis, and thereby may contrib-ute to the carcinogenesis of osteosarcoma (3,4). Binding of Notch ligand to its receptor on the adjacent cells triggers the γ-secretase-mediated proteolytic release of the Notch intracellular domain. The Notch intracellular domain translocates into the nucleus, resulting in the activation of its target genes, such as those in the hairy/enhancer

of split (Hes) and Hes-related with YRPW motif (Hey) families (5). Deregulated Notch receptor expression has been reported in a growing number of human solid tumors, such as pancreas (6), cervix (7) and colon (8), as well as osteosarcoma (9). It has also been reported that manipulation of Notch-1 pathway showed a crucial role for Hes-1 in osteosarcoma invasion and metastasis (4,10). Moreover, blockage of Notch pathway using γ-secretase inhibitor suppressed osteosarcoma growth in vitro and in vivo (3,9), suggesting that the downregulation of Notch may be an effective approach for osteosarcoma therapy.

Recent studies have demonstrated that there is a cross-talk between microRNA (miRNA) and Notch signaling pathways in tumor develop-ment and progression (11). MiRNAs are short, non-coding RNAs that bind to the 3′ untranslated region of cognate messenger RNAs (mRNAs) through fully complementary or imperfect base-pairing repressing the translation or decreasing the stability of the bound mRNAs (12,13). Some miRNAs are reported as oncomirs, which could function either as oncogenes or as tumor suppressors (14). Indeed, miR-21 is signifi-cantly overexpressed in osteosarcoma tissues and knockdown of miR-21 greatly decreases osteosarcoma cell invasion and migration (15). Downregulation of miR-143/145 correlates with the lung metastasis of human osteosarcoma cells and overexpression of miR-143/145 sup-presses cell invasion and angiogenesis (16,17). Upregulation of miR-34a inhibits the proliferation and metastasis of osteosarcoma cells in vitro and in vivo (18). Emerging evidence suggests that miR-34a par-ticipates in the regulation of p53 and Notch pathways consistent with tumor suppressor activity (19,20), suggesting that further mechanis-tic understanding of Notch regulation by miRNAs and finding novel agents that could inactivate Notch would become a promising strategy for the treatment of aggressive osteosarcoma.

Diallyl trisulfide (DATS), a naturally occurring organosulfur com-pound derived from Allium vegetables, is shown to reduce the risk of cardiovascular disease and diabetes, to stimulate immune system, to protect against infections and have antiaging as well as anticancer effects (21). Initial evidence for the anticancer effect of Allium con-stituents is provided by epidemiological studies (22,23). Subsequent laboratory studies showed that Allium constituents can not only offer protection against chemically induced cancer in animal models by alter-ing carcinogen metabolism but also suppress growth of cancer cells in vitro and in vivo by causing cell cycle arrest and apoptosis induction (22,24–26). Our previous studies with osteosarcoma cells demonstrated that DATS could inhibit cell cycle progression and induce apoptosis, as well as reverse drug resistance and lower the ratio of CD133+ cells in conjunction with methotrexate (27,28). DATS is also reported to inhibit angiogenesis by downregulation of vascular endothelial growth factor (VEGF) and affect remodeling of the extracellular matrix through inac-tivation of matrix metalloproteinases (MMPs) (29,30). Recently, we found that DATS treatment concomitantly attenuated Notch-1 expres-sion and upregulated miR-34a in osteosarcoma cells, which led us to conduct this study to determine whether inactivation of Notch-1 could contribute to DATS-induced inhibitory effects and how it is related to specific miRNAs that are typically lost in osteosarcoma.

In this study, we assessed the effect of DATS on cell survival, migration, invasion and angiogenesis, as well as the regulation of Notch-1 by miRNAs in osteosarcoma cells. We also examined the role of Notch-1 in the regulation of cell growth, invasion and angiogenesis, and miRNA expression in osteosarcoma cells and mechanistically investigated the regulation of Notch-1 by small interfering RNA (siRNA) transfection. Most importantly, we determined the role of miR-34a and miR-200b in the regulation of Notch-1, VEGF, MMP-2 and MMP-9 in osteosarcoma cells and their mechanistic correlation with antitumor activity of DATS. Taken together, we conclude that DATS inhibits osteosarcoma growth and aggressiveness by targeting a Notch–miRNA regulatory circuit, which could be a potential novel

Abbreviations: DATS, diallyl trisulfide; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; Hes, hairy/enhancer of split; Hey, Hes-related with YRPW motif; HUVECs, human umbilical vascular endothelial cells; mRNA, messenger RNA; miRNAs, microRNAs; MMPs, matrix met-alloproteinases; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT, reverse transcription; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

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target for the prevention of tumor progression and/or treatment of osteosarcoma.

Materials and methods

Cell culture and experimental reagentsHuman osteosarcoma cell lines U2OS and SaOS-2 (ATCC, Rockville, MD) were cultured in McCoy’s 5A medium, whereas MG-63 (KeyGEN Biotech, Nanjing, China) was maintained in Dulbecco’s modified Eagle’s medium. All media were supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 10 U/ml penicillin G and 100  μg/ml streptomycin. Human umbilical vascular endothelial cells (HUVECs; ATCC) were cultured in F12K medium supplemented with 10% fetal bovine serum, 0.1 mg/ml heparin sulfate and 0.05 mg/ml endothelial cell growth factor (BD Bioscience). All cells were incubated in a 5% CO2 at 37°C. DATS was purchased from Shanghai Hefeng Pharmacy Company (Shanghai, China). Primary antibodies for Notch-1 intra-cellular domain and Hes-1 were purchased from Abcam (Cambridge, UK). Primary antibodies for cyclin D1, VEGF, MMP-2 and MMP-9 were obtained from Cell Signaling Technology (Beverly, MA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and all other chemicals were obtained from Sigma (St Louis, MO), unless otherwise indicated.

Cell growth and survival assayMTT assay was conducted to assess cell growth and survival. Osteosarcoma cells were exposed to different concentrations of DATS (25, 50 and 100 μM) for 1–3 days of treatment, and then MTT assay was carried out as described previously (27). Control cells were treated with 0.1% dimethyl sulfoxide (DMSO) in culture medium.

Cell cycle analysisThe DATS-treated cells, as indicated earlier, were harvested, washed twice in phosphate-buffered saline and then fixed in 70% ethanol for 30 min at 4°C. After washing in cold phosphate-buffered saline thrice, cells were resuspended in 1 ml of phosphate-buffered saline solution with 40 µg of propidium iodide for 30 min at 37°C. Samples were then analyzed for their DNA content by FACS Calibur (Becton Dickinson, San Jose, CA).

Cell migration and invasion assaysCell migration (wound-healing) assay was conducted to determine the capacity of cell migration and invasion as described previously (31). Briefly, the wound was generated when the cells reached around 90% confluency by scratching the surface of the plates with a pipette tip. The cells were incubated in the absence and presence of DATS (25 μM) for 24 and 36 h, respectively, and then photographed at the identical location of the initial image with an Olympus Optical microscope (Melville, NY). The percentages of open spaces covered by migrated cells were determined as described previously (32). The assays were performed in triplicate. In vitro invasion assay was conducted using BD BioCoat Tumor Invasion System (Bedford, MA) according to manufacturer’s protocol (33). Initially, the cells were incubated with different concentrations of DATS (25, 50 and 100  μM) for 24 h. Next, viable cells (2 × 105/200  µl, confirmed by trypan blue exclusion) were seeded in serum-free medium onto the upper chamber, whereas complete medium was added in the lower com-partment as a chemoattractant. After 48 h of incubation, the cells in the upper chamber were removed, and the cells, which invaded through Matrigel matrix membrane, were then fixed and stained with 0.2% w/v crystal violet. Abilities of invasion were quantified by counting the number of invaded cells from six random microscopic fields (×100 magnifications) and represented the average of three independent experiments with duplicate samples.

Matrigel in vitro HUVEC tube formation assayThe 25 μM DATS-treated cells were cultured in serum-free medium for 24 h. The conditioned media were collected, centrifuged and stored at −20°C. HUVECs were trypsinized and seeded (5 × 104 cells per well) in Matrigel-coated well with 250  μl of conditioned medium from osteosarcoma cells treated with DMSO or 25  μM of DATS in serum-free medium for 24 h. VEGF165 (50 ng/ml) was used as a positive control and medium only was used as a negative control. HUVECs grown in growth factor–reduced Matrigel were treated with DMSO or 25 μM of DATS to observe whether DATS directly affected tube formation of HUVECs. The chamber was incubated for 8 h. Five different fields were chosen randomly in each well, and photographs were taken. Image analysis of tubule/capillary length was carried out using Image-Pro Plus software (Media Cybernetics LP, Silver Spring, MD) (34).

Western blot analysisWestern blot analysis was conducted using whole-cell protein lysates of osteo-sarcoma cells. Total cell lysates from different treatment were obtained using

radio immunoprecipitation assay (35) lysis buffer, and western blot was per-formed as described previously (28). The bidimensional optical densities of proteins on the films were quantified and analyzed with Quantity One software (Bio-Rad, Hercules, CA). Values were normalized to the values of β-actin of each sample.

Enzyme-linked immunosorbent assayCells were seeded (1 × 105 cells per well) in six-well plates and treated with 25 or 50 μM of DATS. After 48 h, the cell culture supernatant was harvested and cell count was done after trypsinization. After collection, the supernatant was centrifuged to remove cell debris and stored at −20°C for later VEGF assay using Enzyme-Linked Immunosorbent Assay (ELISA) Kits (R&D Systems, Minneapolis, MN).

Gelatin zymography analysisCells were treated with 50 μM of DATS for 48 h in serum-free medium. The conditioned media were harvested and centrifuged to remove cell debris. The effects of DATS on the gelatinolytic activities of MMP-2 and MMP-9 were examined by gelatin zymography as described previously (36).

Levels of mRNAs by real-time reverse transcription–PCRTo determine the mRNA expression of Notch-1, Hes-1, Hey-1, Hey-2, VEGF, MMP-2 and MMP-9 mRNAs, 2 μg of total RNAs from each sam-ple were used for reverse transcription (RT) reaction using a RT system (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was conducted using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA) and performed with SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) in triplicate. Data were analyzed using Ct method and normalized by glyceraldehyde 3-phosphate dehydroge-nase expression in each sample. Sequences of PCR primers were described previously (37).

MiRNAs expression analysisTo determine the expression of miRNAs (miR-21, miR-34a, miR-143, miR-145 and miR-200b/c) in osteosarcoma cells, All-in-One miRNA qRT–PCR Detection Kit (GeneCopoeia, Rockville, MD) was used following manufac-turer’s protocol. Briefly, the extracted RNA was reverse-transcribed in the presence of a poly-A polymerase with an oligo-deoxythymidine adaptor. Quantitative PCR was then carried out with SYBR Green detection as stated earlier (38). Data were analyzed using Ct method and normalized by RNU6B expression in each sample.

Transfection of Notch-1 siRNA and complementary DNAThe Notch-1 siRNA and Notch-1 ICN complementary DNA plasmid encoding the Notch-1 intracellular domain were prepared as described previously (37). The sequences of Notch-1 siRNA were as follows: NS 1# 5′-UACAGUACUGA CCUGUCCACUCUGG-3′ and NS 2# 5′-CCGCCU UUGUGCUUCUGUUCU UCGU-3′. Briefly, U2OS cells were seeded (2 × 105 cells per well) in six-well plates overnight and transfected with Notch-1 siRNA or scramble con-trol siRNA by Lipofectamine 2000. U2OS cells were stably transfected with human Notch-1 ICN or vector alone and maintained under G418 selection. After 48 h of transfection, the cells were collected for cell survival and invasion assay, tube formation assay, ELISA assay, gelatin zymography analysis, west-ern blot analysis and real-time RT–PCR of miRNAs as described previously.

Transfection of miR-34a and miR-200b mimicsU2OS cells were seeded in six-well plates overnight and transfected with miRNA mimics or miRNA-negative control (Ambion) using Lipofectamine 2000 as described previously (39). After 3 days of transfection, the transfected cells were harvested for western blot analysis and real-time RT–PCR of miR-NAs and mRNAs were performed as described previously.

Statistical analysisData were expressed as mean ± SD from three independent experiments. Statistical analyses were carried out using the Student’s t-test with GraphPad StatMate software (GraphPad Software, San Diego, CA). P  <  0.05 was considered statistically significant.

Results

DATS inhibited osteosarcoma cell survivalWe determined the effect of DATS on osteosarcoma cell proliferation in a variety of cultured cell lines (U2OS, SaOS-2 and MG-63 cells). As assessed by MTT assay, the treatment of osteosarcoma cells for 1–3 days with 25, 50 or 100  μM of DATS resulted in cell growth inhibition. Moreover, the inhibition of cell growth was found to be dose- and time-dependent in U2OS, SaOS-2 and MG-63 cells (Figure 1A). To further

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investigate the effect of DATS on cell survival in more detail, we con-ducted a cell cycle analysis using U2OS, SaOS-2 and MG-63 cells treated with 25 or 50 μM of DATS for 48 h. As shown in Figure 1B, DATS-treated osteosarcoma cells were arrested at G0/G1 phase. Furthermore, the higher the concentration of DATS used, the more U2OS, SaOS-2 and MG-63 cells were blocked at G0/G1 phase. These data suggest that DATS could elicit pronounced cell growth-inhibitory effect on osteosarcoma cells, which is consistent with our previously published reports (27).

DATS decreased osteosarcoma cell migration and invasionTo examine the effect of DATS on osteosarcoma cell migration and invasion, we conducted cell migration (wound-healing) assay and in vitro invasion assay using U2OS, SaOS-2 and MG-63 cells. We found that DATS treatment significantly decreased the capacity of wound healing in U2OS, SaOS-2 and MG-63 cells compared with those cells without DATS treatment (Figure 2A). We also found that DATS markedly inhibited the capacity of tumor invasion by a con-centration-dependent manner in U2OS and SaOS-2 cells (Figure 2B).

These results demonstrate that DATS could suppress osteosarcoma cell migration and invasion.

Reduced tube formation of HUVECs induced by conditioned media from DATS-treated osteosarcoma cellsTo determine whether conditioned media from osteosarcoma cells could induce the tube formation of HUVECs and whether condi-tioned media from DATS-treated osteosarcoma cells could reduce the tube formation, we performed the tube formation assay in growth factor reduced-Matrigel in vitro. Conditioned media from U2OS, SaOS-2 and MG-63 cells (Figure 2C) as well as from VEGF (Figure  2D) were able to significantly induce the tube formation of HUVECs in 8 h incubation (compared with Figure 2D, medium only as negative control). Importantly, we found that conditioned media from U2OS, SaOS-2 and MG-63 cells treated with 25  μM of DATS inhibited tube formation compared with the medium from cells treated with DMSO (Figure 2C, control). To exclude the pos-sibility that inhibition of tube formation by conditioned media from

Fig. 1. (A) DATS decreased cell survival of U2OS, SaOS-2 and MG-63 cells in a dose- and time-dependent manner as assessed by MTT assay. Results represented the mean ± SD of three individual experiments having six determinations per experiment for each experimental condition. *P < 0.05. (B) DATS treatment for 48 h caused G0/G1 cell cycle arrest in U2OS, SaOS-2 and MG-63 cells as measured by flow cytometry analysis. Results were representative of three independent experiments. Data were plotted as the percentage of cells in each cell cycle phase.

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25  μM DATS-treated osteosarcoma cells was mediated through direct effect of DATS on tube formation, HUVECs grown in growth factor–reduced Matrigel were treated with 25 μM of DATS for 8 h (Figure  2D). DATS had no effect on tube formation within 8 h of incubation (compared with Figure 2D, DMSO control). These results

suggest that DATS could exert pronounced angiogenesis-inhibitory effect on osteosarcoma cells.

To directly determine whether the reduced tube formation was a consequence of the decreased secretion of VEGF, we analyzed the levels of VEGF in conditioned media from U2OS, SaOS-2 and

Fig. 2. DATS decreased wound-healing capacity (A) and cell invasion (B) in U2OS, SaOS-2 and MG-63 cells, as well as reduced the HUVECs tube formation (C and D) and the VEGF secretion from U2OS, SaOS-2 and MG-63 cells (E). The DATS-treated cells were collected for wound-healing assay, in vitro invasion assay and conditioned media from DATS-treated cells for tube formation, ELISA assay as described in the Materials and methods. Migration abilities were determined as the percentages of open spaces covered by migrated cells. Invasion abilities were quantified by counting the number of invaded cells from six random microscopic fields (×100 magnifications) and represented the mean ± SD of three independent experiments. Quantification of angiogenic activity was calculated by measuring the tubule/capillary length using Image-Pro Plus software. Results were representative of three independent experiments. *P < 0.05. **P < 0.01.

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MG-63 cells treated with DATS. As shown in Figure 2E, 25 μM of DATS treatment markedly decreased the VEGF secretion from osteo-sarcoma cells compared with DMSO control (P < 0.05).

Effect of DATS on Notch-1, Hes-1, VEGF, MMP-2 and MMP-9 in osteosarcoma cellsTo further understand the molecular mechanism involved in DATS-induced proliferation inhibition, alterations in the cell survival Notch pathway were investigated. Notch-1 and its target gene Hes-1, Hey-1, Hey-2 expression in U2OS, SaOS-2 and MG-63 cells treated with 50  μM of DATS for 48 h were assessed using real-time RT–PCR analysis. Compared with control, there was a reduction of Notch-1, Hes-1, Hey-1 and Hey-2 mRNA levels after DATS treatment, suggest-ing that DATS resulted in the transcriptional inactivation of Notch-1 pathway in osteosarcoma cells (Figure 3A). It is important to note that the active functional form of Notch is Notch-1 intracellular domain. Therefore, we focused our studies on Notch-1 intracellular domain at the protein level and Notch-1 in all figure legends means active functional form of Notch-1. In agreement with the real-time RT–PCR data, western blot showed that Notch-1 protein levels were downregu-lated in DATS-treated U2OS, SaOS-2 and MG-63 cells (Figure 3B). Together, we concluded that DATS was effective in inhibiting the transcription and translation of the Notch-1 gene. To confirm the inac-tivation of Notch-1 signaling by DATS, we also detected the other

Notch downstream genes such as Hes-1 and cyclin D1. Indeed, DATS treatment inhibited Hes-1 and cyclin D1 expression (Figure 3B).

Notch-1 pathway was reported to play crucial roles in osteosar-coma invasion and metastasis (4). Notch-1 was also reported to cross-talk with VEGF and MMPs, which were critically involved in the processes of tumor cell invasion and metastasis (40–42). To explore whether the downstream effect of DATS induced by downregulation of Notch-1 was associated mechanistically with VEGF and MMPs reduction, we detected the alterations in the expression of VEGF, MMP-2 and MMP-9. Compared with control cells, the mRNA and protein expression of VEGF, MMP-2 and MMP-9 were dramatically reduced in DATS-treated U2OS, SaOS-2 and MG-63 cells (Figure 3A and B). Next, we examined whether DATS could lead to a decrease in MMPs activities. Gelatin zymography analysis demonstrated that both MMP-2 and MMP-9 activities were reduced in DATS-treated cells (Figure 3C). Most importantly, we also found that DATS could lead to a decrease in the levels of VEGF secreted in the culture medium as assessed by ELISA assay (Figure 3D).

Effect of DATS on the expression of miRNAs in osteosarcoma cellsWe examined the effect of DATS on the expression of miRNAs (miR-21, miR-34a, miR-143, miR-145 and miR-200b/c) in osteosarcoma cells. The results revealed that DATS treatment increased the relative expressions of miR-143 and miR-145, whereas decreased the relative

Fig. 3. DATS downregulated the expression of Notch-1, Hes-1, Hey-1, Hey-2, VEGF, MMP-2 and MMP-9 mRNA (A), Notch-1, Hes-1, cyclin D1, VEGF, MMP-2 and MMP-9 protein (B), as well as decreased the activities of MMP-2, MMP-9 (C) and VEGF (D) in U2OS, SaOS-2 and MG-63 cells. Real-time RT–PCR, western blot, gelatin zymography analysis and ELISA assay were performed as described in the Materials and methods. Glyceraldehyde 3-phosphate dehydrogenase was used to normalize the mRNA level and results were presented as the percentage relative to control (cells treated with DMSO). β-actin was used as a sample loading control. Data were expressed as mean ± SD from three independent experiments.

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expression of miR-21 in U2OS, SaOS-2 and MG-63 cells (Figure 4A). DATS treatment increased the relative expression of miR-34a in U2OS cells but not in SaOS-2 and MG-63 cells (Figure 4A). Interestingly, miR-200b and miR-200c expressions were very low in U2OS and MG-63 cells and DATS was able to upregulate their expression (Figure 4A). These data suggest that DATS differentially regulates the expression of these tumor-associated miRNAs in osteosarcoma cells.

Effect of Notch-1 deficiency on cell survival, invasion and angio-genesis in osteosarcoma cellsTo confirm the role of Notch-1 in proliferation, invasion and angio-genesis of osteosarcoma cells, we conducted a gene knockdown experiment in U2OS cells. We found that Notch-1 siRNA transfection resulted in reduced expression of Notch-1, Hes-1 and cyclin D1, but most interestingly, the knockdown of Notch-1 led to decreased expres-sion of VEGF, MMP-2 and MMP-9 (Figure 5A). The downregulation of Notch-1 resulted in decreased cell growth and invasion as well as decreased the HUVECs tube formation (Figure 5B–D). Combination of Notch-1 siRNA and 25 μM DATS treatment further inhibits pro-liferation, invasion and angiogenesis in U2OS cells (Figure 5B–D). Western blot analysis showed that Notch-1 siRNA plus DATS induced inactivation of Notch-1 activity to a greater degree, compared with DATS alone (Figure 5E). The downregulation of Notch-1 by siRNA also promoted DATS-induced decrease in the activities of VEGF, MMP-2 and MMP-9 (Figure 5F).

Subsequently, we investigated the effect of Notch-1 overexpression on DATS-induced proliferation and invasion inhibition. Notch-1 complementary DNA-transfected U2OS cells were treated with 50 µM DATS for 48 h. Compared with negative control, Notch-1 protein levels were upregulated in Notch-1 complementary DNA-transfected cells (Supplementary Figure 1A, available at Carcinogenesis Online). Moreover, the upregulation of Notch-1 resulted in a significant

increase in cell growth and invasion, and this upregulation alleviated DATS-induced cell growth and invasion inhibition to a certain degree (Supplementary Figure 1B and C, available at Carcinogenesis Online). These results provided evidence for a potential role of Notch-1 during DATS-induced proliferation and invasion inhibition.

Effect of Notch-1 siRNA on the expression of miRNAs in osteosarcoma cellsWe next explored the effect of Notch-1 inactivation by siRNA on the expression of the tumor-associated miRNAs (miR-21, miR-34a, miR-143, miR-145 and miR-200b/c) in U2OS cells. We found that Notch-1 downregulation led to reexpression of miR-34a, miR-143, miR-145, miR-200b and miR-200c, which are typically lost in osteo-sarcoma cells as well as resulted in a decrease in miR-21 expression (Figure  4B), and these results are consistent with DATS treatment results as presented previously in Figure 4A.

The role of miR-34a and miR-200b in the regulation of Notch-1, VEGF, MMP-2 and MMP-9 in osteosarcoma cellsTo investigate the mechanistic role of miR-34a and miR-200b in the regulation of Notch-1, VEGF, MMP-2 and MMP-9 in osteosarcoma cells, we conducted transfection experiment using U2OS cells by miR-34a or miR-200b mimics. As expected, the transfection of miR-34a and miR-200b mimics increased the relative levels of miR-34a and miR-200b, respectively, which consequently decreased the relative levels of Notch-1 protein in U2OS cells (Figure 6A). Reexpression of miR-34a and miR-200b also inhibited the expression and activities of VEGF, MMP-2 and MMP-9 in U2OS cells (Figure  6A and B). These data suggest that miR-34a and miR-200b play key roles in the regulation of many factors that are involved in the development and progression of osteosarcoma, such as Notch-1, VEGF, MMP-2 and MMP-9.

Fig. 4. DATS upregulated miR-34a, miR-143, miR-145 and miR-200b/c expression, whereas downregulated miR-21 expression in U2OS, SaOS-2 and MG-63 cells (A). Notch-1 siRNA increased the relative expression of miR-34a, miR-143, miR-145, miR-200b/c and decreased the relative expression of miR-21 in U2OS cells (B). Real-time RT–PCR analysis of miRNAs was performed as described in the Materials and methods. RNU6B was used to normalize the mRNA level and results were presented as the percentage relative to control (A: Cells treated with DMSO; B: Cells transfected with control siRNA). Data were expressed as mean ± SD from three independent experiments.

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Effect of reexpression of miR-34a and miR-200b on cell survival, invasion and angiogenesis in osteosarcoma cellsWe further examine the role of miR-34a and miR-200b in the growth and metastasis of osteosarcoma cells by transfection of mimics into U2OS cells. We found that reexpression of miR-34a and miR-200b significantly decreased the cell survival and invasion of U2OS cells as well as reduced the HUVECs tube formation (Figure 6C), indicating that restoration of miR-34a and miR-200b is mechanistically associated with the regulation of cell survival, invasion and angiogenesis in osteosarcoma cell and thus DATS-mediated reexpression of miR-34a and miR-200b is mechanistically associated with its biologic effects on the growth and metastasis.

Discussion

Accumulating in vitro and in vivo studies have shown that DATS could exert its antitumor effect in a variety of malignancies, includ-ing prostate, stomach, lung, breast, cervix and colon cancer (22–26). DATS is known to suppress the proliferation of various cancer cells by inducing apoptosis and cell cycle arrest (24,25). In this study, we showed that DATS elicited a dramatic effect on proliferation inhi-bition and G0/G1 cell cycle arrest in U2OS, SaOS-2 and MG-63

osteosarcoma cells. Moreover, we found that DATS inhibited osteo-sarcoma cell migration, invasion and angiogenesis. To elucidate the molecular mechanisms of proliferation and aggressiveness inhibition by DATS, we investigated the activity of Notch-1 signaling pathway, which plays a key role in the process of cell survival (3,6,9), invasion and metastasis (4,10).

In this study, we demonstrated for the first time that DATS downreg-ulated the transcription and translation of Notch-1 and its downstream genes, Hes-1, Hey-1, Hey-2 and cyclin D1, resulting in osteosarcoma cell growth inhibition. It is now well accepted that aberrant activation of Notch-1 signaling promotes a myriad of cellular activities such as cell survival, proliferation, migration, invasion and angiogenesis as well as suppresses apoptosis through a growth factor-mediated sur-vival pathway in many human malignancies, including osteosarcoma (3,4,9,10). Therefore, DATS-induced cell survival inhibition could be partly mediated via inactivation of Notch-1 activity. This was fur-ther confirmed by the combination of Notch-1 siRNA and DATS, which further inhibited osteosarcoma cell proliferation. Similarly, the combination of DATS with Notch-1 siRNA further downregulated Notch-1 and Hes-1 expression. Therefore, these data indicate that together with DATS treatment, downregulation of Notch-1 by siRNA could be a possible therapeutic approach for better management of osteosarcoma growth.

Fig. 5. Notch-1 siRNA decreased the expression of Notch-1, Hes-1, cyclin D1, VEGF, MMP-2 and MMP-9 protein (A), cell growth (B) and invasion (C) in U2OS cells and the HUVECs tube formation (D). Notch-1 siRNA-transfected U2OS cells were treated with 25 μM DATS for 24 h. Combination of Notch-1 siRNA and DATS treatment further inhibited proliferation (B), invasion (C), angiogenesis (D) and reduced Notch-1, Hes-1 expression (E) and VEGF, MMP-2 and MMP-9 activities (F) in U2OS cells. CS, control siRNA; NS, Notch-1 siRNA; DATS, 25 μM DATS; NS + DATS, Notch-1 siRNA + 25 μM DATS. The transfected cells were collected for cell survival and invasion assay, tube formation assay, western blot, gelatin zymography analysis and ELISA assay as described in the Materials and methods. β-actin was used as a sample loading control. Invasion abilities were quantified by counting the number of invaded cells from six random microscopic fields (×100 magnifications) and represented the mean ± SD of three independent experiments. Quantification of angiogenic activity was calculated by measuring the tubule/capillary length using Image-Pro Plus software. Results were representative of three independent experiments. *P < 0.05 relatively control siRNA. **P < 0.05 relatively Notch-1 siRNA or DATS.

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Aberrant Notch-1 signaling has been shown to be associated with metastatic phenotype and to regulate the expression of a variety of important genes in some cellular responses, including metastasis-related genes such as VEGF, MMP-2 and MMP-9 (4,10,40–42). Our previous studies have demonstrated that Notch-1 inactivation by curcumin could be mechanistically associated with the downregulation of MMP-2 and MMP-9, resulting in osteosarcoma cell invasion inhibition (37). In this study, we also found that DATS inhibited the expression and activities of MMP-2 and MMP-9 as well as VEGF in U2OS, SaOS-2 and MG-63 osteosarcoma cells. VEGF and MMPs

are crucial in the processes of tumor cell invasion, angiogenesis and metastasis, and VEGF, MMP-2 and MMP-9 are directly linked with angiogenesis and degradation of the basement membrane collagen leading to metastasis (43,44). Indeed, our results showed that DATS inhibited migration and invasion of osteosarcoma cells through the Matrigel and reduced the tube formation of HUVECs. These results are consistent with the inactivation of VEGF, MMP-2 and MMP-9 by the downregulation of Notch-1, resulting in the inhibition of osteosarcoma cell invasion and angiogenesis. Moreover, the downregulation of Notch-1 by siRNA transfection promoted DATS-induced invasion

Fig. 6. Reexpression of miR-34a and miR-200b decreased the expression of Notch-1, VEGF, MMP-2 and MMP-9 protein (A) and the activities of MMP-2, MMP-9 and VEGF (B) and reduced cell survival and invasion in U2OS cells as well as the HUVECs tube formation (C). The transfection of miR-34a or miR-200b mimics was conducted in U2OS cells and western blot, gelatin zymography analysis, ELISA assay, cell survival and invasion assay, tube formation assay were performed as described in the Materials and methods. RNU6B was used to normalize the mRNA level and results were presented as the percentage relative to control (cells transfected with miRNA-negative control). β-actin was used as a sample loading control. Invasion abilities were quantified by counting the number of invaded cells from six random microscopic fields (×100 magnifications) and represented the mean ± SD of three independent experiments. Quantification of angiogenic activity was calculated by measuring the tubule/capillary length using Image-Pro Plus software. Results were representative of three independent experiments. *P < 0.05 relatively control miRNA.

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and angiogenesis inhibition, associated with further downregulation of VEGF, MMP-2 and MMP-9. Therefore, our data clearly suggest that Notch-1 signaling cross-talks with VEGF and MMPs during DATS-induced cell invasion and angiogenesis inhibition. Our results also provide a possibility that the downregulation of Notch-1 by DATS could suppress osteosarcoma cell invasion and angiogenesis partly through the downregulation of VEGF, MMP-2 and MMP-9.

As stated previously, we have shown for the first time that DATS could exert its antitumor activity by targeting Notch-1 in osteosarcoma cells and further investigated how DATS downregulates Notch-1 sign-aling as well as the expression of its downstream genes. To that end, it is known that miRNAs are key players, which function as endogenous post-transcriptional gene regulators to mediate protein synthesis or mRNA stability (12,13). A  large number of miRNAs are reported to be associated with tumor development and progression by regu-lation of the expression/transcription of many tumor-related genes (14,45,46). Several miRNAs upregulate significantly and specifically miR-21 is aberrantly overexpressed in many solid tumors, which is involved in tumor progression, poor survival and reduced thera-peutic effects (47). Indeed, miR-21 is significantly overexpressed in osteosarcoma, and the suppression of miR-21 decreased the invasion and migration in MG-63 cells (15). Moreover, reversion- inducing cysteine-rich protein with Kazal motifs (RECK) is found to be a direct target that is negatively regulated by miR-21 in osteosarcoma cells and human osteosarcoma samples (15), and it inhibits the invasion of osteosarcoma cells by decreasing the activity of MMPs (48). A recent study has shown that Notch-1 upregulation is associated with increased expression of miR-21, suggesting that miR-21 may play a role in the regulation of Notch-1 signaling (49). Our findings showed that inactivation of Notch-1 led to decreased miR-21 expression, and thus DATS-mediated downregulation of miR-21 expression could be responsible for the biologic effects of DATS in osteosarcoma cells.

Emerging evidence demonstrates that miR-143 and miR-145, poten-tial tumor suppressors, could modulate vascular smooth muscle cells differentiation by inactivation of Notch-1 signaling (50), whose expres-sion has been linked with tumor invasion and metastasis (4,10). Reduced expression of miR-143 and miR-145 has been reported in several cancers, such as colorectal, prostate, ovarian, gastric cancer and osteosarcoma (16,17,45,51). Reexpression of miR-143 or miR-145 in osteosarcoma cells causes downregulation of MMP-13 or VEGF, resulting in the inhibi-tion of tumor invasion and metastasis (16,17). Additionally, the restoration of miR-143 reduces osteosarcoma cell viability and induces apoptosis via an antiapoptotic molecule, BCL-2 (51). Therefore, targeting miR-143 and miR-145 is likely to have beneficial effects toward designing strate-gies for the prevention of tumor progression and/or therapy for osteosar-coma. Here, our data showed that Notch-1 downregulation could increase the expression of miR-143 and miR-145, which is consistent with DATS-mediated reexpression of miR-143 and miR-145 associated with the inhibition of cell proliferation, invasion and angiogenesis, all of which is partially due to inactivation of Notch-1 and its downstream genes.

Another important tumor suppressor miRNA is miR-34a, which has been found to participate in p53 and Notch pathways regulation (19,20). It has been reported that the expression of miR-34a is lower or undetectable in pancreatic, breast, non-small cell lung cancer and osteosarcoma (11,52,53). It has been also reported that miR-34a induced G1 arrest and apoptosis via their targets, CDK6, E2F3, cyclin E2 and BCL-2, in a p53-dependent manner in osteosarcoma cells (52). Moreover, transfection of miR-34a to glioma cells is shown to downregulate the protein expression of Notch-1, Notch-2 and CDK6 (20). Restoration of miR-34a expression in the pancreatic cancer cells is suggested to downregulate Notch-1 and Notch-2, indicating that Notch-1 and Notch-2 may be downstream genes of miR-34a (54). Our results demonstrated that DATS treatment upregulated miR-34a expression in U2OS cells and Notch-1 downregulation by siRNA also increased its expression. However, we did not observe these effects in SaOS-2 and MG-63 cells. One possible reason for the lack of miR-34a upregulation could be that tumor suppressor protein p53 is known to induce miR-34a expression thereby leading cells to apoptosis. U2OS cells with

wild-type p53 show high expressed p53 protein, whereas SaOS-2 and MG-63 cells have p53 mutations resulting in accumulation of mutant, non-functional p53 protein (52), and thus increased miR-34a expression in response to DATS or Notch-1 siRNA can be observed only in U2OS cells. Furthermore, we found that reexpression of miR-34a resulted in the inhibition of cell proliferation, invasion and angiogenesis, associated with concomitant attenuation of Notch-1 and downregulation of its downstream genes, such as VEGF, MMP-2 and MMP-9. These data demonstrate that miR-34a may be involved in DATS-induced inhibition of tumor development and progression, potentially via the direct modulation of downstream target Notch-1.

Recent studies have suggested that miR-200 serve as a potential tumor suppressor, primarily by repressing the acquisition of epithe-lial-to-mesenchymal transition phenotype during tumor develop-ment and progression (55,56). Decreased expression of miR-200 has been observed in many tumors such as pancreatic, breast and pros-tate cancer, which is associated with tumor invasion and metastasis (57). Our study demonstrated for the first time that miR-200b and miR-200c were downregulated in osteosarcoma cells, and Notch-1 inactivation led to increased expression of miR-200b and miR-200c, suggesting that Notch-1 could be involved in the regulation of miR-200b and miR-200c and DATS could lead to the reexpres-sion of miR-200b and miR-200c, which would be highly valuable in reverting tumor cell aggressiveness. We also found that reexpres-sion of miR-200b inhibited osteosarcoma cell growth, invasion and angiogenesis. Furthermore, reexpression of miR-200b or miR-34a led to the downregulation in the expression of Notch-1 and that inactivation of Notch-1 resulted in the upregulation of miR-200b or miR-34a in osteosarcoma cells, showing a negative mutual feedback loop of Notch-1 and miRNAs. These findings are also consistent with treatment of cells with DATS, suggesting that miRNAs that are lost in osteosarcoma cells could be reexpressed using our novel agent DATS.

Collectively, we presented experimental evidence, which strongly suggests that DATS could be useful for inhibiting osteosarcoma development and progression via suppression of Notch-1 signaling, accompanied by downregulation of Hes-1, VEGF, MMP-2 and MMP-9, as well as upregulation of specific tumor-suppressive miRNAs (miR-34a, miR-143, miR-145 and miR-200b/c). Therefore, our data will result in better treatment outcome of patients diagnosed with osteosarcoma, which requires further in-depth preclinical experiments using relevant animal models.

Supplementary material

Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/

Funding

National Natural Science Foundation of China (30973018 and 81172551); Shandong Technological Development Project of China (2011GGH21831).

Conflict of Interest Statement: None declared.

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Received June 16, 2012; revised November 9, 2012; accepted February 10, 2013

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