Tomatidine Represses Invasion and Migration of Human Osteosarcoma U2OS and HOS Cells by Suppression of Presenilin 1 and c-Raf–MEK–ERK Pathway

Osteosarcoma, which is the most prevalent malignant bone tumor, is responsible for the great majority of bone cancer-associated deaths because of its highly metastatic potential. Although tomatidine is suggested to serve as a chemosensitizer in multidrug-resistant tumors, the anti-metastatic effect of tomatidine in osteosarcoma is still unknown. Here, we tested the hypothesis that tomatidine suppresses migration and invasion, features that are associated with metastatic process in human osteosarcoma cells and also investigate its underlying pathway. Tomatidine, up to 100 μM, without cytotoxicity, inhibited the invasion and migration capabilities of human osteosarcoma U2OS and HOS cells and repressed presenilin 1 (PS-1) expression of U2OS cells. After the knockdown of PS-1, U2OS and HOS cells’ biological behaviors of cellular invasion and migratory potential were significantly reduced. While tomatidine significantly decreased the phosphorylation of c-Raf, mitogen/extracellular signal-regulated kinase (MEK), and extracellular signal-regulated protein kinase (ERK)1/2 in U2OS cells, no obvious influences on p-Jun N-terminal kinase, p38, and Akt, including their phosphorylation, were observed. In ERK 1 silencing U2 OS cells, tomatidine further enhanced the decrease of their migratory potential and invasive activities. We conclude that both PS-1 derived from U2OS and HOS cells and the c-Raf–MEK–ERK pathway contribute to cellular invasion and migration and tomatidine could inhibit the phenomenons. These findings indicate that tomatidine might be a potential candidate for anti-metastasis treatment of human osteosarcoma.

tomatidine affects the invasion and migration of human osteosarcoma cells and attempted to define its underlying mechanisms.

Cytotoxicity of Tomatidine in Osteosarcoma U2OS and HOS Cells
For the cell viability experiment, a microculture tetrazolium (MTT) (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was performed to determine the cytotoxicity of tomatidine. After 24 h of treatment, the viability of osteosarcoma U2OS and HOS cells in the presence of concentrations of 25, 50, 75, and 100 µM of tomatidine was not significantly different to that of the controls (0 µM) in MTT assay (U2OS: p = 0894; HOS: p = 0.136) ( Figure 1). Thus, a 24-h treatment with tomatidine up to 100 µM had no cytotoxic effect on U2OS and HOS cells. We used this concentration range for tomatidine in all subsequent experiments to investigate its anti-metastatic properties.

Tomatidine Represses U2OS and HOS Cells Migration and Invasiveness
We used a modified Boyden chamber migration and invasion assays to test the effect of tomatidine on invasive properties of U2OS and HOS cells in vitro. After treating for 24 h, the Boyden chamber assay without Matrigel showed that tomatidine significantly dose-dependently reduced the migratory potential in U2OS and HOS cells (U2OS: p < 0.001; HOS: p < 0.001) ( Figure 2). The modified Boyden chamber assay with Matrigel also showed that tomatidine dose-dependently reduced the invasive activity in U2OS and HOS cells (U2OS: p < 0.001; HOS: p < 0.001).

Tomatidine Reduces PS-1 Expression of U2OS Cells
We employed the protease array, which showed repression of PS-1 secretion in U2OS cells after treatment of 100 µM tomatidine for 24 h, to identify the underlying mechanism of the anti-metastatic actions of tomatidine in osteosarcoma cells, ( Figure 3A). However, no significant effects on MMP-2 and nine secretions were observed in the protease array. We subsequently performed the western blot analysis to validate the finding in the protease array and found that 100 µM of tomatidine significantly repressed the PS-1 protein expression of U2OS cells (p = 0.001) ( Figure 3B).

PS-1 Knockdown Reduces Migration and Invasion of U2OS and HOS Cells
We transformed cells with a small interfering RNA (siRNA) targeting PS-1 expression for 24 h and measured the protein expression and the mRNA level in western blotting and reverse transcription-polymerase chain reaction (RT-PCR), respectively, to further confirm whether reduction of PS-1 interferes with migratory potential and invasive activity of U2OS and HOS cells (U2OS: protein: p < 0.001 and RNA: p < 0.001; HOS: protein: p < 0.001 and RNA: p = 0.002) ( Figure 4A). Subsequently, we performed Boyden chamber migration and modified Matrigel invasion assays while using siRNA of PS-1 for 24 h and 48 h to compare the amount of migratory and invasive cells, respectively. Unsurprisingly, the knockdown of PS-1 significantly decreased the migratory potential and invasive activities of U2OS and HOS cells (U2OS: migration: p = 0.008 and invasion: p = 0.034; HOS: migration: p = 0.001; and, invasion: p < 0.001) ( Figure 4B).

Tomatidine Reduces the c-Raf-MEK-ERK Pathway in U2OS Cells
Western blotting was employed to further investigate the molecular mechanisms since MAPKs and PI3K pathways may be dependent signaling of PS-1. In the analysis, c-Raf, mitogen/extracellular signal-regulated kinase (MEK), MAPKs, and PI3K-Akt pathways were detected in U2OS cells. As a result, tomatidine decreased the phosphorylation of c-Raf, MEK, and ERK 1/2 in U2OS cells, but no obvious influence on JNK 1/2, p38, and Akt, including their phosphorylation, was observed ( Figure 5).

Tomatidine Inhibits Cellular Migration and Invasion in ERK 1 Knockdown U2OS Cells
We conducted siRNA directly against the ERK 1 with and without treatment of 100 µM tomatidine to identify whether the ERK pathway interferes with migratory potential and invasive activities in U2OS cells, and performed Boyden chamber migration and modified Matrigel invasion assays to compare the amount of migratory and invasive cells. Predictably, the knockdown of ERK 1 significantly decreased the migratory potential and invasive activities in U2OS cells (p < 0.05 and p < 0.05, respectively) and tomatidine further enhanced the decrease of migratory potential and invasive activities in ERK 1 silencing U2OS cells (p < 0.05 and p < 0.05, respectively) ( Figure 6). However, with and without treatment of 100 µM tomatidine, ERK 1 knockdown could not further enhance the decrease of PS-1 expression (data not shown), which implies that the c-Raf-MEK-ERK pathway might be not the upstream signaling of PS-1.

Discussion
In the study, tomatidine, without cytotoxicity, attenuated migratory potential and invasiveness of U2OS and HOS cells. Although MMP-2 and MMP-9 are key enzymes and they contribute to the process of osteosarcoma cell invasion and metastasis in our previous research [35][36][37][38], there were no effects of tomatidine on MMP-2 and nine secretions of U2OS cells in the protease array. Intriguingly, the repression of PS-1 in U2OS cells was observed after treatment of 100 µM tomatidine and the tomatidine's repression of PS-1 protein expression was verified in western blotting. The silencing of PS-1 confirmed the anti-metastatic properties of migration and invasion of U2OS and HOS cells by PS-1. Through a further analysis of MAPKs and the PI3K pathways, tomatidine decreased the phosphorylation of c-Raf, MEK, and ERK 1/2 in U2OS and HOS cells, whereas there was no evident influence on JNK 1/2, p38, and Akt, and their phosphorylation. Furthermore, the decrease of migratory potential and invasive activities, which was caused by the ERK 1 knockdown in U2OS cells, was enhanced by tomatidine. These results implied that tomatidine's inhibition of invasion and migration in human osteosarcoma U2OS and HOS cells resulted from the attenuation of PS-1 and the c-Raf-MEK-ERK pathway, rather than JNK, p38, and PI3K-Akt signaling.
PS homologs PS-1 and PS-2 participate in several signaling pathways that regulate cell survival and tumorigenesis. PS-1 mutant overexpression has been reported to induce cell apoptosis [39], while the loss of PS-1 and mutant PS-1 mice have higher skin and carcinogen-induced brain tumorigenesis, respectively [24,40]. PS-1 promotes tumor invasion and metastasis of gastric cancer both in vitro and in vivo, in addition to the positive correlation with lymph node metastasis and the poor overall survival rate [25]. Conversely, the γ-secretase inhibitor DAPT inhibits gastric cancer cell growth and EMT and the results of the treatment are consistent with the outcomes of treatment with PS-1 silencing [25,26]. The therapeutic effect of γ-secretase inhibition was also observed in lung cancer by the derepression of DUSP1 and inhibition of ERK [27]. In the present study, we found that tomatidine represses PS-1 to inhibit the biological behaviors of migration and invasion in U2OS and HOS cells, which indicates that PS-1 might represent a novel prognostic biomarker and a potential therapeutic target for anti-metastasis treatment of osteosarcoma. Moreover, notch signaling regulates osteosarcoma proliferation and migration through ERK phosphorylation, so PS might be the upstream signaling of the ERK pathway and the inhibition of PS can lead to ERK activation [41]. However, in the study, the silencing of ERK 1 seemed not to affect PS-1 expression, which suggests that the c-Raf-MEK-ERK pathway might be not the upstream signaling of PS-1. While the c-Raf-MEK-ERK pathway and PS-1 pathway both simultaneously contribute to invasion and migration of U2OS and HOS cells, they might be independently or the c-Raf-MEK-ERK pathway might be the downstream signaling of PS-1. Hence, further tests are required to make it explicitly clear. Anyway, PS-1 and the c-Raf-MEK-ERK pathways both actually affect the invasion and migration of U2OS and HOS cells.
The diverse MAPK members and PI3K/Akt are activated in response to various extracellular stimuli and have distinct downstream targets, including cell motility, migration, invasion, proteinase-induction, and angiogenesis, which all contribute to metastasis [42]. Besides, ERK 1/2 and JNK are thought to play a central role in regulating the expression of MMPs to implicate cell migration and proteinase-induction [16,17,42]. Tomatine, which is a secondary metabolite from tomato, suppresses MMP-2 and MMP-9 activities and cell proliferation in breast cancer MCF-7 cell line and structure-activity relationships of α-, β 1 -, γ-, and δ-tomatine and tomatidine against various cancer cells have been studied [29,43]. Alpha-tomatine inactivates PI3K/Akt and ERK signaling pathways and nuclear factor (NF)-κB and AP-1 binding activities to inhibit the invasion and migration of human lung adenocarcinoma A549 cells by reducing u-PA MMP-2 and MMP-9 [18]. However, the invasion and migration of human non-small cell lung cancer NCI-H460 cells are suppressed by α-tomatine through inactivating the focal adhesion kinase/PI3K/Akt signaling pathway, which reduces the binding activity of nuclear factor (NF)-κB and downregulates the MMP-7 expression [44].
Of particular interest is that tomatidine inhibits iNOS and cyclooxygenase-2 expressions to display the anti-inflammatory effect through the suppression of NF-κB and JNK pathways in LPS-stimulated mouse macrophages [45]. In addition to anti-inflammatory, anti-tumorigenic, and lipid-lowering activities [45,46], tomatidine has been suggested to serve as a chemosensitizer in combination chemotherapy, which uses chemotherapeutic drugs for the treatment of multidrug-resistant cancers [47]. Moreover, tomatidine inhibits the invasion of human lung adenocarcinoma A549 cells through the suppression of ERK and Akt pathways and MMP-2 and 9 expressions [34]. However, in the study, tomatidine's inhibitory properties of migration and invasion in U2OS and HOS cells are induced by the suppression of the c-Raf-MEK-ERK 1/2 pathway and the repression of PS-1 secretion, but that has no effect on MMP-2 and 9. These findings reveal a unique concept of pathway and direction for tomatidine in anti-metastatic therapy of osteosarcoma. In future, the determination of therapeutic potential and pharmacodynamics properties of tomatidine on osteosarcoma metastasis in vivo is imperative.

Cell culture and Tomatidine Treatment
Being obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan), the human osteosarcoma U2OS (15-yr-old female) cells and HOS (13-yr-old female) cells were supplemented with 10% FBS and 1% penicillin/streptomycin and then cultured in DMEM and Eagle's MEM, respectively. The cell cultures were maintained at 37 • C in a humidified atmosphere of a 5% CO2 incubator. Tomatidine was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell Migration and Invasion Assays
After treatment with the indicated concentrations of tomatidine (0, 25, 50, 75, and 100 µM), the cells were seeded into the upper section of the Boyden chamber (Neuro Probe, Cabin John, MD, USA) without or with Matrigel at densities of 2.0 × 10 5 /mL for the U2OS cells and HOS cells, and then incubated at 37 • C for 24 h, respectively. Finally, the migratory cells in the Boyden chamber migration assay and invasive cells in the modified Boyden chamber invasion assay were counted under a light microscope, as described previously [17,48].

Protease Array Analysis
A protease array (35 proteases) analysis was used to evaluate the protein lysates from vehicle-or 100 µM tomatidine-treated cells, according to the manufacturer's protocols (Human Protease Array Kit, Catalog Number ARY021B, R&D Systems, Minneapolis, MN).
As described previously, blots were then incubated with a horseradish peroxidase goat anti-rabbit or anti-mouse IgG for 1 h and the intensity of each band was measured by densitometry [17,37,48].

Small Interfering RNA
For silencing PS-1 protein expression, a unique siRNA inhibiting human PS-1 (s111) and negative-control siRNA (4390844) were purchased from Applied Biosystems Instruments (Foster City, CA, USA). For silencing the ERK1 protein expression, a unique siRNA inhibiting human ERK1 (SC-29307) and negative-control siRNA (SC-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 5 × 10 5 U2OS cells and HOS cells were grown in 6 cm cell culture dishes overnight. A total of 150 pmol of PS-1 siRNA was transfected into the cells while using lipofectamine RNAiMAX transfection reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The silencer negative control siRNA, a nonsense siRNA duplex, was used as a control.

Statistical Analysis
For all of the measurements, analysis of variance was followed by one-way analysis of variance (ANOVA) with post hoc Turkey's HSD tests for more than two groups with equal sample sizes per group. When two groups were compared, the data were analyzed whileusing Student's t-test. Each experiment was performed in triplicate and three independent experiments were performed. p values < 0.05 was considered to be statistically significant.

Conclusions
In conclusion, U2OS and HOS cells-derived PS-1 and the c-Raf-MEK-ERK signaling pathway, not JNK, p38 and PI3K/Akt signaling, may both contribute to cellular invasion and migration. This phenomenon of PS-1 s repression of invasion and migration in U2OS and HOS cells could be activated by tomatidine. Certainly, our work reinforces the idea that tomatidine possesses the suggestive behaviors of anti-metastatic properties in human osteosarcoma cells, which contributes to a better understanding of the mechanism that is responsible for these effects.