2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol reverses EGF-induced cell migration and invasion through down-regulation of MDM2 in breast cancer cell lines

ABSTRACT

2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ), a novel synthesized combretastatin A-4
(CA-4) analogue, is identified as a microtubule inhibitor and has been shown to exert anticancer activity in breast cancer cells. Here, we found that SQ reversed epidermal growth factor (EGF)-induced motility and invasion in breast cancer cell lines by the in vitro Wound healing and Transwell assay. Further studies showed that SQ treatment resulted in inhibitory alteration of EGF-stimulated epithelial-to-mesenchymal transition (EMT) and MMP-2 activity. What is more, SQ significantly inhibited the EGF-induced mouse double minute 2- (MDM2) expression and transcription factor Twist1 expression. In addition, compared with the control cells, MDM2 overexpression up-regulated Twist1 expression and dramatically promoted cell migration and invasion, MDM2 under-expression also down-regulated Twist1 expression and suppressed cell motility and invasion. Taken together, our findings suggest that the inhibitory effects of SQ on migration and invasion were related to the suppression of MDM2 and Twist1 signal axis.

Keywords:

• Combretastatin A-4 analogue
• EMT
• MDM2
• Twist1
• migration
• invasion

Introduction

Breast cancer is a common cause of death in women’s cancers at all ages. The main reason of cancer death results from metastasis instead of the primary tumor itself.^1,2 Cancer metastasis includes a series of closely related steps: epithelial-to-mesenchymal transition (EMT), cell motility, local invasion, intravasation, traveling through the blood or lymphatic circulation, extravasation, and transiting in secondary organs.^3–5

Breast cancer cells with EMT phenotype lose their epithelial characteristics and escape from the primary tumor.^6,7 Multiple pathways including epidermal growth factor (EGF), integrin, Wnt and nuclear factor (NF)-κB pathways have been found to trigger EMT. The main feature of the EMT pathways is that the activated transcription factors (Snail1, Slug and Twist1) initiate EMT program in the epithelial cells, down-regulating E-cadherin and other epithelial cell phenotype-associated genes and activating the mesenchymal cell phenotype-related genes, such as N-cadherin and Vimentin.⁸ Some studies proved that EGF or the overexpression of transcription factor Twist1 can induce the process of EMT in breast cancer cells.^9–13 As reported previously, EGF played a leading role in promoting breast cancer metastasis, probably by activating extracellular signal-regulated kinae 1/2 (ERK 1/2) or phosphoinositide-3 kinase/Akt (PI3K/Akt) signal pathway.^11,14,15 Transcription factor Twist1 is involved in the cancer invasiveness and metastasis process.^16,17 Twist1 regulates EMT-related proteins, such as down-regulation of E-cadherin and up-regulation of Vimentin and matrix metalloproteinases (MMPs), driving the breast cancer cells toward metastasis ¹². Furthermore, MMPs including MMP-2 and MMP-9 play an essential role in cancer metastasis by degradation of the extracellular matrix (ECM).^18–20

Mouse double minute 2 (MDM2), an oncogene which is usually highly expressed in malignancies including solid tumors, sarcomas and leukemias, regulates the growth in breast cancers.^21,22 Recently, some studies have demonstrated that MDM2 overexpression was related to cancer invasiveness and metastasis.²³ In recent studies, MDM2 enhances cell mobility and invasion through MDM2-mediated Slug degradation, causes its E3 ubiquitin ligase activity.^24,25 In another study, MDM2 promotes migration and invasion through a p53 independent manner instead of depending on its ability as an E3 ubiquitin ligase. However, the exact role of MDM2 in cell metastasis is still unclear.²⁶ Therefore, it is important to find key molecules and molecular mechanisms which regulate breast cancer metastasis and explore new promising therapeutic targets.

Combretastatins are isolated from the South African willow tree Combretum caffrum. Among all combretastatins, Combretastatin A-4 (CA-4) is the most effective with tubulin-binding ability and cytotoxicity (Figure 1a).²⁷ CA-4 is also a vascular-disrupting agent (VDA)^28–30 and has an anti-metastatic activity.³¹ However, CA-4 is unstable and easily isomerized to inactive form. SQ is a novel synthesized CA-4 analogue which hybridizes with organoselenium specifically (Figure 1b).³² In our previous study, SQ has been shown as a promising microtubule destabilizing agent, which can induce apoptosis in human breast cancer cells through p53-independent manner. Besides, SQ directly induces MDM2 degradation through an ubiquitin-proteasome system. Moreover, the apoptosis induced by SQ is associated with the inhibition of MDM2.^32,33 In this current study, the effect of SQ on cell migration and invasion and its underlying mechanisms were investigated. We found that SQ suppressed EGF-induced invasion and motility in breast cancer cells by inhibiting MDM2 and down-regulating Twist1 expression.

Figure 1. The chemical structure of combretastatin A-4 (CA-4) and SQ.

Materials and methods

Reagents

Compound SQ was synthesized and purified by our group as described previously.³³ 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 4ʹ,6-diamidino-2-phenylindole (DAPI) and EGF were purchased from Sigma-Aldrich (St. Louis, MO, USA). Crystal violet and Lipo6000 tansfection reagent were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against MDM2, GAPDH, HRP-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). E-Cadherin, N-cadherin, Vimentin, FAK, MMP-2, MMP-9, p53, Snail1, Slug and Twist1 antibodies were obtained from Proteintech Group (Chicago, IL, USA).

Cell culture

Human breast cancer cells MCF-7 and MDA-MB-231 were obtained from American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% streptomycin and 1% penicillin at 37 ºC in humidified incubator under an atmosphere with 5% CO₂. MCF-7 and MDA-MB-231 cells were tested monthly for mycoplasma by the GMyc-PCR Mycoplasma Test assay (Yeasen Biotech, Shanghai), and all the cell lines using in our experiment are negative.

Cell viability assay

Cell viability was assessed by the MTT assay. This assay was performed as described previously.³³

Small interfering RNA (sirna)

MDM2 siRNA #1 (5ʹ-GCCACAAAUCUGAUAGUAU-3ʹ) and MDM2 siRNA #2 (5ʹ-AGAUCCUGCUGCUUUCGCAUU-3ʹ); scrambled siRNA #1 (5ʹ-GGACGCAUCCUUCUUAAUU-3ʹ) and scrambled siRNA #2 (5ʹ-UUCUUCGAACGUGUCACGUTT-3ʹ) were synthesized by GenePharma (Suzhou, China). Cells were cultured at 50% confluency and transfected with siRNA using Lipo6000 reagent followed the manufacturer’s procedure.

Plasmid construction and transfection

The expression vector for human MDM2 (pGEX4T2-MDM2) was obtained from Addgene (Cambridge, USA). Full-length cDNA of MDM2 was generated by PCR amplification with the primers derived from human MDM2 (5ʹ-CCGGAATTCGCCACCATGTGCAATACCAACAT-3ʹ and 5ʹ-CGCGGATCCCGGGGGAAATAAGTTAGCAC-3ʹ). The PCR products with BamHI/EcoRI were subcloned into eukaryotic pcDNA3.1 expression vector. The resulted MDM2 plasmid was verified by DNA sequencing. For stable transfection, the empty vector or MDM2 plasmid was transfected into MCF-7 cells with Lipo6000 Reagent. After transfected for 24 h, G418 (Life technologies, MA USA) was added to select stable transfected cell lines.

Cell adhesion assay

Cells were pretreated with EGF or the combination with SQ (25 nM and 50 nM) for 24 h and then plated at 2 × 10⁴ per well of collagen I coated 96-well plates (Becton Dickinson, Bedford, USA), followed by incubation for 30 min. Adherent cells were fixed with in 4% paraformaldehyde and stained with 0.2% crystal violet. Then the adherent cells were counted and imaged with an inverted microscope (Olympus IX51).

Wound healing assay

Cells were seeded in 6-well plates and generated confluent cell monolayers. The wound was made across the monolayer by a sterilized pipette tip. Wound closure was measured at 0 and 24 h within scrape line and photographed by an inverted microscope. Quantification of relative wound healing area was calculated using the image J software.

Cell migration and invasion assay

2 × 10⁴ cells suspended in 200 μL serum-free medium were seeded into the upper chamber of Transwell insert (Corning, MA, USA) for the migration assay, while the upper chamber was coated with 50 μl matrix gel for the invasion assay. The plates were incubated for 24 h for migration and 48 h for invasion, the membranes of chamber were then fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Cells were counted and imaged with an inverted microscope.

Quantitative real-time PCR

MDM2 mRNA level was quantified by real-time PCR as described previously ³³. The primer sequences were as follows: MDM2, 5ʹ-ATTGGTTGTCTCATACTGG-3ʹ and 5ʹ-CTCAACACAAGCTGAAGAGG-3ʹ; GAPDH, 5ʹ-CAATGACCCCTTCATTGACC-3ʹ and 5ʹ-TGGAAGATGGTGATGGGATT-3ʹ. GAPDH was set as a control.

Immunofluorescence

Cells were cultured on a 6-well plate with cover glass, then treated with EGF or the combination with SQ (25 nM) for 24 h. Immunofluorescence staining was performed as described previously to determine E-cadherin and Vimentin.³³

Gelatin zymography

Cells were seeded in 6-well plates and then treated with EGF or EGF combined with SQ for 24 h. The culture supernatants were collected and centrifuged to remove cells and debris. Equal amount of samples were run in 7.5% SDS-polyacrylamide gels containing gelatin. After electrophoresis, the gels were washed and incubated with reaction buffer at 37 ̊C for 48 h. The gels were next stained in 0.5% Coomassie Brilliant Blue solution and destained in 40% methanol and 10% acetic acid solution.

Western blot

Western blot was determined as described previously.³³ Densitometry analysis was performed using Image J software.

Statistical analysis

Data values were shown as mean ± SD from three independent experiments. Statistical differences were assessed by one-way ANOVA using SPSS software 16.0, as appropriate. P < 0.05 was considered statistically significant.

Results

SQ inhibits EGF-induced EMT

It has been shown that EGF is one of the potent breast cancer motility factors and it can promote metastasis.^11,34 In this study, cellular morphological changes of MCF-7 cells under EGF treatment were investigated in the presence or absence of SQ. As shown in Figure 2a, cells formed a typical compact cell-to-cell contact in the control cells. While cells acquire obvious mesenchymal phenotype changes after EGF treatment for 24 h, such as losing cell-to-cell contact, increasing cell scattering and elongating spindle shape. However, SQ (25 nM and 50 nM) treatment mitigated EGF-induced mesenchymal phenotype changes, and the cells exhibited a classical epithelial-like morphology. We also found that EGF increased the adhesion ability, and this increased adhesion ability could be suppressed by SQ (Figure 2b). Moreover, SQ at the concentration of 25 nM and 50 nM has no significant influence on cell proliferation under EGF-treated conditions (Figure 2c). In addition, EGF also caused to cell-to-cell contact decrease and enhanced cell scattering in MDA-MB-231 cells. However, SQ could inhibit cell scattering and mesenchymal phenotype changes at the doses of 5 and 10 nM that do not affect cell proliferation under EGF treatment in MDA-MB-231 cells (supplementary Figure 1a and b). Taken together, these morphological changes indicate the efficacy of SQ in inhibiting EGF-induced EMT. Then, we examined the expressions of EMT-related markers to further clarify whether SQ inhibited EGF-induced EMT. As shown in Figure 2d and supplementary Figure 1c, the expression level of epithelial marker E-cadherin was decreased, the expression levels of mesenchymal marker N-cadherin and Vimentin were increased when cells were treated with EGF in MCF-7 and MDA-MB-231 cells. Moreover, SQ treatment significantly reversed EGF-induced down-regulation of E-cadherin and suppressed EGF-induced the expression level increase of N-cadherin and Vimentin (Figure 2d and supplementary Figure 1c) in MCF-7 and MDA-MB-231 cells. In addition, Focal adhesion kinase (FAK), a critical mediator of integrin and growth factor receptor signaling pathway, promotes cancer cell adhesion, invasion and metastasis.³⁵ Results from western blot (Figure 2d) showed that the treatment with SQ could attenuate EGF-induced FAK expression in MCF-7 cells. Furthermore, the immunofluorescence analysis proved that SQ significantly reversed EGF-downregulated E-cadherin expression increase (Figure 2e) and decreased the EGF-induced Vimentin expression increase (Figure 2f). These results demonstrate that EGF induces the occurrence of EMT, and SQ reverses the progress of EGF-induced EMT.

Figure 2. SQ inhibits EGF-induced EMT in MCF-7 cells. (A) Effect of SQ on morphological changes of EGF-induced cells. Cells were treated with EGF (100 ng/ml) or EGF (100 ng/ml) plus SQ (25 or 50 nM) for 24 h. Scale bar = 50 μm. (B) Effect of SQ on EGF-induced adhesion. (C) Effects of SQ on cell viability. Cells were treated with SQ (25 or 50 nM), EGF (100 ng/ml) alone or the combination with SQ at the concentration of 25 or 50 nM for 24 h. (D) EMT-related proteins were determined by western blot. (E, F) Immunofluorescence analysis of E-cadherin and Vimentin. Scale bar = 50 μm. *P < 0.05, ** P < 0.01 vs control; ^# P < 0.05, ^## P < 0.01 vs the cells only treated with 100 ng/ml EGF.(A).

SQ inhibits EGF-induced migration and invasion

As EMT contributes to tumor cells acquiring migrative and invasive properties,^4–6 we determined the effects of SQ on EGF-induced motility and invasion in breast cancer cells. In the wound healing assay, EGF (100 ng/ml) promoted cell motility when compared with the control. Importantly, SQ effectively suppressed EGF-induced motility in MCF-7 and MDA-MB-231 cells (Figure 3a and supplementary Figure 1d). Moreover, inhibitory effects of SQ on the EGF-induced invasion and migration were detected by the transwell assay. As illustrated in Figure 3b, 3c, supplementary Figure 1e and 1f, under the treatment with EGF, migrative and invasive cells were significantly increased. Notably, SQ reversed the EGF-induced motility and invasion significantly in MCF-7 and MDA-MB-231 cells. Since EMT-related transcription factors such as Snail1, Slug, and Twist1 are usually activated in the process of migration and invasion,^8,36 we determined the key EMT-transcriptional inducers to investigate the potential molecular mechanism of SQ on EGF-induced invasion and migration. Our study found that EGF significantly enhanced the expression of Twist1, and Twist1 expression was decreased by the treatment with SQ (Figure 3d and supplementary Figure 1c). However, the expressions of Snail1 and Slug had almost no change after EGF or EGF plus SQ treatment (Figure 3d). In addition, MMP-2 and MMP-9 are involved in metastasis through the degradation of ECM, which could be regulated by Twist1 signaling.^10,12 As a result, western blot and gelatin zymography were performed to assess the protein levels and activities of MMP-2 and MMP-9 in EGF-induced cells. The results showed that SQ inhibited both the protein expression level and activity of EGF-induced MMP-2. However, EGF-induced MMP-9 level was not significantly affected by SQ treatment (Figure 3e and 3f).

Figure 3. SQ inhibits EGF-induced migration and invasion in MCF-7 cells. (A) Cell motility was assessed by wound healing assay. Quantitative analyses of the percentage of wound closure at 24 h compared with 0 h were shown on the right. (B, C) Cell migrative and invasive capacities were determined by the transwell assay. Representative images of migrative and invasive cells were shown on the left and the quantitative analyses of migrative and invasive cells per field were shown on the right. (D) Western blots showed the expressions of Snail1, Slug and Twist1. (E) The expression levels of MMP-2 and MMP-9 were detected by western blot. (F) MMP-2 and MMP-9 activities were evaluated by gelatin zymography. * P < 0.05, ** P < 0.01 vs control; ^# P < 0.05, ^## P < 0.01 vs the cells only treated with 100 ng/ml EGF.

SQ inhibits egf-induced MDM2 expression

Recent studies have suggested the linkage between MDM2 expression and the increasing potential of motility, invasion and metastasis.^26,37 In our previous studies, SQ inhibited MDM2 expression by a p53-independent manner.^32,33 We hypothesized that the regulation of MDM2 was involved in the inhibitory effect of SQ on EGF-induced migration and invasion. As we suspected, the protein and mRNA levels of MDM2 were obviously increased in the present of EGF analyzed by western blot and real-time PCR. However, SQ significantly suppressed EGF-induced MDM2 expression increase (Figure 4a and 4b). In addition, EGF also increased the protein and mRNA levels of MDM2 in another breast cancer cell line MDA-MB-231. Furthermore, SQ at the concentration of 5 nM and 10 nM inhibited EGF-induced high expression of MDM2 in MDA-MB-231 cells (Figure 4c and 4d). In addition, the total p53 expression was decreased in the presence of EGF, and SQ could not reverse the total p53 expression significantly (supplementary Figure 2).

Figure 4. SQ inhibits EGF-induced expression of MDM2. (A, B) The effects of SQ on EGF-induced MDM2 expression increase were assessed by western blot and real-time PCR in MCF-7 cells. (C, D) The protein and mRNA levels of MDM2 were determined by western blot and real-time PCR in MDA-MB-231 cells. * P < 0.05, ** P < 0.01 vs control; ^# P < 0.05, ^## P < 0.01 vs the cells only treated with 100 ng/ml EGF.

MDM2 overexpression promotes cell migration and invasion

To further confirm our hypothesis, the empty vector and MDM2 plasmid were transfected into MCF-7 cells. The increased protein expression of MDM2 in the stable MDM2-overexpressing cells was shown in Figure 5a. Since we found that Twist1 was participated in the inhibitory effects of SQ on EGF-induced migration and invasion (Figure 3d), we explored the expression of Twsit1 in MDM2-overexpressing cells. The western blot results showed that Twist1 expression was dramatically enhanced followed by MDM2 overexpression (Figure 5b). Additionally, SQ (25 and 50 nM) decreased the expression of MDM2 and Twist1 significantly in MDM2-overexpressing cells. Moreover, wound healing assay showed that MDM2 overexpression promoted cell motility compared with the control. However, SQ (25 and 50 nM) significantly suppressed cell motility in MDM2-overexpressing cells (Figure 5c). Moreover, the effects of MDM2 overexpression on invasion and migration were also determined by the transwell assays. In stable transfected cell lines, MDM2 overexpression promoted migration and invasion (Figure 5d and 5e) compared with the control group. However, SQ (25 and 50 nM) significantly reduced migration and invasion (Figure 5d and 5e) in MDM2-overexpressing cells.

Figure 5. Up-regulation of MDM2 promotes migration and invasion. (A) Western blot was performed to detect the expression of MDM2 in MCF-7 cells stably transfected with MDM2 plasmid and the empty vector. (B) The MDM2-overexpressing cells were treated with SQ (25 and 50 nM) for 24 h. MDM2 and Twist1 expression levels were detected by western blot. (C) Cell motility was determined by wound healing assay. (D, E) Migrated and invasive cells were analyzed by transwell assay. * P < 0.05, ** P < 0.01 vs the empty vector; ^# P < 0.05, ^## P < 0.01 vs MDM2 plasmid.

Effect of knockdown MDM2 on migration and invasion

To further explore the role of MDM2 in invasion and migration, MCF-7 cells were transfected with MDM2 siRNA. The decreased expression of MDM2 was proved by western blot (Figure 6a and supplementary Figure 3a). Furthermore, in the presence of EGF, the expression of MDM2 was down-regulated by siRNA transfection, which resulted in reduction of Twist1 expression (Figure 6b and supplementary Figure 3b). Down-regulation of MDM2 and Twist1 expression through the treatment with SQ in siRNA tranfected cells were more significant compared with MDM2 siRNA transfected cells in presence of EGF (Figure 6b and supplementary Figure 3b). For the wound healing assay, the delayed wound closure was detected in cells transfected with MDM2 siRNA compared with scrambled siRNA in the presence of EGF (Figure 6c and supplementary Figure 3c). The transwell assay was then performed to investigate migration and invasion in siRNA-transfected cell lines. As shown in Figure 6d, Figure 6e, supplementary Figure 3d and 3e, the migrated and invasive cells were decreased in MDM2 siRNA-transfected cells compared with scrambled siRNA-transfected cells in the presence of EGF. Moreover, the addition of SQ significantly inhibited cell motility and invasion in EGF co-existed MDM2 siRNA-transfected cells. Taken together, our data suggests that MDM2 significantly regulated the expression of Twist1, which was involved in the inhibitory effect of SQ on EGF-induced migration and invasion.

Figure 6. Effect of MDM2 knockdown on migration and invasion. (A) Western blot analysis for the expression of MDM2 in MCF-7 cells transfected with MDM2 siRNA and scrambled siRNA. (B) MCF-7 Cells were transfected with MDM2 siRNA for 24 h, and incubated with SQ (25 nM) for another 24 h in the presence of EGF (100 ng/ml). The expression levels of MDM2 and Twist1 were analyzed by western blot. (C) Wound closure area was photographed and measured. (D, E) Migrated and invasive cells were analyzed by transwell assay. * P < 0.05, ** P < 0.01 vs scrambled siRNA.

Discussion

Cancer metastasis is the most common cause of the death in breast cancers.^1,2 Several studies have elucidated that EGF or a combination of other growth factors can induce EMT in breast cancer cells.^10,34 The current study found that EGF stimulated motility and invasion, characterized by the regulation of EMT-related markers such as E-cadherin, N-cadherin, Vimentin and increase of the expression levels of MMP-2/9 and FAK in MCF-7 cells. Moreover, EGF significantly enhanced the expression of a EMT-related key transcriptional factor Twist1, but not Snail and Slug. In addition, we also found that EGF promoted cell migration and invasion in MDA-MB-231 cells, accompanied by up-regulation of N-cadherin and Vimentin expressions, and down-regulation of E-cadherin expression. Besides, the transcriptional factor Twist1 was also enhanced by EGF treatment in MDA-MB-231 cells.

MDM2 is an oncogene that was first cloned in a murine double-minute chromosome in a tumorigenic mouse 3T3DM cells.³⁸ MDM2 contains a N-terminal p53-binding domain and a ring finger domain which possesses E3 ligase activity, catalyzing the proteasomal degradation of p53 to promote tumor malignancy.^37,39–43 Here, we found that EGF strongly increased the expression of MDM2 at the mRNA and protein levels in human breast MCF-7 and MDA-MB-231 cells. Meanwhile, we demonstrated that the overexpression of MDM2 can significantly increase the capability of cell invasion and migration. Furthermore, the down-regulation of MDM2 after transfection with MDM2 siRNA reduced the movement and invasive ability compared with the control under the treatment with EGF.

Some studies indicate that the expression of transcription factor Twist1 in breast cancers could significantly enhance EMT, cell invasion and metastasis.^{12,13,44–48} In addition, several studies have shown that Twist1 enhanced MDM2-mediated p53 degradation, which aggravated the malignancy of human sarcoma cells.^49,50 Recent study has shown that the expression of MDM2 enhanced migration and invasiveness in cells which were independent of the intact MDM2 ring-finger domain and p53.²⁶ In this study, MDM2 tended to induce the transcription factor Twist1 expression, which was demonstrated by overexpression and silencing of endogenous MDM2, thereby affected cell motility and invasion. However, the relationship between MDM2 and Twist1 requires further study.

Our previous work has demonstrated that SQ, a novel microtubule-destabilizing agent, strongly suppressed tumor proliferation and induced apoptosis, which was associated with MDM2 inhibition.^32,33 In this study, we further identified that SQ reversed EGF-induced invasion and migration through the inhibition of MDM2 expression in MCF-7 and MDA-MB-231 cells. In addition, the treatment of SQ significantly inhibited EGF-enhanced key EMT-transcriptional factor Twist1 expression, but had almost no effect on Snail and Slug expression. Taken together, SQ might down-regulate Twist1 through inhibiting MDM2, and ultimately suppress EGF-induced motility and invasion. On the other hand, it was reported that Erk or PI3K signaling may play a role in EGF-induced breast cancer metastasis.^11,14,15 As a result, we cannot rule out the involvement of Erk or PI3K signaling in the present study, which will be investigated in the future study. Interestingly, SQ inhibited the high MMP-2 expression induced by EGF, but had little effect on MMP-9 expression increase, which indicates that MMP-2 might be involved in the inhibition of cell migration and invasion induced by SQ treatment in MCF-7 cells.

In summary, our study suggests that SQ inhibited EGF-induced migration and invasion by down-regulating the expressions of MDM2 and EMT-related proteins. MDM2-regulated Twist1 signaling pathway might be involved in the inhibitory effects of SQ on migration and invasion, which suggests that SQ is a potential anti-cancer metastasis agent for breast cancer therapy (Figure 7). These results also provide further evidence for the development of compound SQ as a pharmacological agent. Meanwhile, the axis of MDM2/Twist1 signaling could be a potential drug target for breast cancer treatment.

Figure 7. Diagram of the proposed molecular mechanisms by which SQ inhibits EGF-induced migration and invasion.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by grants from the National Natural Science Foundation of China (81872394 and 8167130571), Young and middle age backbone personnel training program of Shenyang Pharmaceutical University (ZQN2015003) and Liaoning BaiQianWan Talents Program (2016921065).