Isoliquiritigenin attenuates the invasive capacity of breast cancer cells via up-regulating the tumor suppressor RECK

Abstract

Breast cancer is the primary cause of cancer-related deaths in women worldwide, mainly due to tumor cell metastasis. Isoliquiritigenin (ISL), a dietary compound extracted from licorice, has been well studied for its anti-oxidant and anti-inflammatory activity, however its anti-tumor invasive capacity remains largely unknown. The RECK gene acts as a tumor suppressor due to its repressive effect on tumor cell invasion. To date, no studies have determined if ISL can induce the expression of RECK. Here, we found that ISL could efficiently attenuate the invasive capacity of breast cancer cells via up-regulating RECK protein level and down-regulating micro RNA-21 (miR-21) in a dose- and time-dependent manner. Furthermore, we firstly reported that miR-21 inhibited the expression of RECK via translation repression rather than mRNA degradation in breast cancer cells. In breast cancer cell lines MDA-MB-231 and Hs-578T exposed to ISL, restored miR-21 expression by miR-21 mimics reversed the inhibitory and inductive effects of ISL on cellular invasiveness and RECK expression, respectively. Thus, the repression of miR-21 and subsequent induction of RECK may be the anti-tumor mechanism of ISL, at least partly. The results suggest an alternative for dietary tumor intervention.

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1. Introduction

Breast carcinoma is the major cause of cancer-related deaths in women worldwide, leading to more than 500 000 deaths in 2012.¹ The current breast cancer treatments are less desirable due to cancer metastasis. Studies have attempted to determine the underlying mechanisms of tumor metastasis, whereby carcinoma cells can form a solid tumor in another distant organ.² However, the underlying mechanisms within metastasis remain largely ambiguous due to multiple metastatic pathways. Thus, it is essential and urgent to elucidate the molecular mechanisms of metastasis.

The reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene inducing cell flat morphology was isolated by Takahashi et al. It encodes a glycoprotein about 110 kDa with multiple epidermal growth factor-like and serine-protease inhibitor-like domains. Previously, it has been proved that RECK down-regulation is frequent in many types of cancer-derived cell lines, suggesting that it may act as a tumor suppressor during carcinogenesis.³ RECK expression can be attenuated by several oncogenes, such as RAS and HER2.^4–6 Furthermore, restoration of RECK expression in malignant cells resulted in inhibition of the invasive activity and tumor-induced angiogenesis via extracellular matrix regulation through repressing gelatinases, a subclass of matrix metalloproteinases (MMPs), namely MMP2 and MMP9.^3,7 It is reasonable to hypothesize that any agent which could enhance RECK gene expression could have clinical use in the treatment of cancer.

The microRNAs (miRNAs) are a class of endogenous noncoding RNA molecules that repress target gene expression via binding to site(s) within the 3′ untranslated region (UTR) of specific mRNAs resulting in either translation suppression or less frequent mRNAs degradation.⁸ It has been well revealed that deregulation of miRNAs expression plays a vital role in human cancers.⁹ Furthermore, human miRNA genes are frequently located at fragile sites and genomic regions involved in cancers.¹⁰ Studies have suggested the involvement of miRNAs in many cancer cellular processes, such as cellular invasion.¹¹ In initiation and development of cancer, miRNAs may act as either tumor promoters or suppressors via repression of anti-oncogenes or oncogenes.

Dietary compounds have been well documented for their anti-tumor effects.¹² For example, a study about curcumin, a phytochemical component isolated from the spice turmeric, showed that it could act as a new anti-cancer drug proved by preclinical and clinical studies.¹³ Previously, we also found glabridin (GLA), a phytochemical isolated from licorice, may possess some anti-tumor potentials, such as anti-stem cell like properties¹⁴ and anti-angiogenisis¹⁵ in breast cancer. Additionally, the dietary compound isoliquiritigenin (ISL), another phytochemicals extracted from licorice, has been well studied for its anti-oxidant¹⁶ and anti-inflammatory effects.¹⁷ Much attention also has been paid on its anti-tumor effects. Jianping Chen et al. proved the inhibitory effect of ISL on the angiogenesis of breast cancer via promoting hypoxia inducible factor-1α (HIF-1α) proteasome degradation and interacting with VEGFR-2 to block its kinase activity.¹⁸ In addition, ISL could cause lung cancer cell growth inhibition and apoptosis by targeting both wild type and L858R/T790M mutant EGFR.¹⁹ Decreased JNK/AP-1 signaling was also proved the involvement in the repressive effect of ISL on the migration and invasion of prostate cancer cells.²⁰ In a more recent study, inactivated PI3K/Akt pathway, the phosphorylation of p38 and NF-κB DNA binding activity-induced inhibition of MMP2 and MMP9 may be responsible for the repressive effect of ISL on the migration and invasion of breast cancer cell MDA-MB-231.²¹ In the present research, we reported that ISL showed anti-tumor effects via down-regulating miR-21 expression, followed by the induction of RECK protein and subsequent inhibition of MMP9. Our data added novel understandings on the anti-tumor effects of ISL. These results indicated that ISL, a compound isolated from dietary supplements, may exert ideal anti-tumor effects and could provide an alternative or additive for the treatment of breast cancer.

2. Materials and methods

2.1 Cell culture and reagents

Human breast cancer cell lines, MDA-MB-231 and Hs-578T were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured according to the ATCC's recommendations. MDA-MB-231 cells were cultured in L-15 medium; whereas Hs-578T cells were cultured in Dulbecco's Modified Eagle's medium (DMEM; Life Technologies/Gibco, Grand Island, NY, USA). Both of the medium contained 10% fetal bovine serum (FBS; Life Technologies/Gibco), 100 mg mL⁻¹ streptomycin, and 100 U mL⁻¹ penicillin (Life Technologies/Gibco). MDA-MB-231 cells were maintained in an incubator without CO₂ at 37 °C; Hs-578T cells were grown in the presence of 5% CO₂. ISL, dissolved in dimethyl sulfoxide (DMSO), was obtained from Sigma-Aldrich (St. Louis, MO, USA), was >98% pure, and was stored at −20 °C. All the other reagents used in our present study were of analytical grade or the highest grade available.

2.2 Determination of cell viability

The human breast cancer cell lines MDA-MB-231 and Hs-578T (2 × 10⁴ cells) were seeded in 96 well plates for 24 h. The cells were treated with 0.0, 5.0, 10.0, 20.0, or 40.0 μM ISL for 24 h or 48 h. Subsequently, cells were incubated with 20 μL Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay solution for 4 h. The absorbance at 450 nm was measured by using a multi-well plate reader (Model 680; Bio-Rad, Hercules, CA, USA). Non-treated cells served as controls.

2.3 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from cells receiving different treatments was isolated using Trizol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. For miR-21 detection, total RNA (1 μg) was reverse transcribed into cDNA using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) with miRNA-specific looped reverse primers. Forward and reverse primers were purchased from RiboBio (Guangzhou, China). The reverse transcription reaction conditions were as follows: 42 °C for 15 min and 85 °C for 5 s. The U6 snRNA acted as the loading control. The qRT-PCR assays were performed with the Applied Biosystems 7300 Sequence Detection System (Applied Biosystems) for 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 70 °C for 30 s. For RECK detection, total RNA (2 μg) was reverse transcribed into cDNA with AMV Reverse Transcriptase (Promega, Madison, WI, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The primers used in our study are listed in Table 1. Another qRT-PCR was performed using the Applied Biosystems 7300 Sequence Detection System (Applied Biosystems) with cycling conditions as follows: 95 °C for 15 s and 60 °C for 1 min for 40 cycles. The two types of qRT-PCRs mentioned above were carried out with Power SYBR® Green master mix (Applied Biosystems). Fold changes in expression of each gene were calculated by a comparative threshold cycle (C_t) method with the formula 2^−(ΔΔC_t).

Table 1 Sequences of the primers used for real time RT-PCR

RECK	Forward	5′-AGCAACCGAGCCCGTATGT-3′
	Reverse	5′-CCGAGTAGGCAGCACACACA-3′
GAPDH	Forward	5′-GTCAGTGGTGGACCTGACCT-3′
	Reverse	5′-AGGGGAGATTCAGTGTGGTG-3′

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2.4 Reverse-transcriptase polymerase chain reaction (RT-PCR)

Total cellular RNA was isolated with Trizol® (Invitrogen) according to the manufacturer's protocols. RNA (2 μg) was transcribed into cDNA using AMV Reverse Transcriptase (Promega, Madison, WI, USA). Primers used are listed in Table 2. The reactions were determined by analyzing the PCR products on 2% (w/v) agarose gels. GAPDH product was applied as a cDNA loading control.

Table 2 Sequences of the primers used for RT-PCR

MMP2	Forward	5′-GCATGGCGATGGATA-3′
	Reverse	5′-GGAAGCGGAATGGAAAC-3′
MMP9	Forward	5′-CACCTCTGCCCTCACCATGAGC-3′
	Reverse	5′-CATGGTGAGGGCAGAGGTGTCT-3′
GAPDH	Forward	5′-GTCAGTGGTGGACCTGACCT-3′
	Reverse	5′-AGGGGAGATTCAGTGTGGTG-3′

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2.5 Western blotting

Cell lysis solution was prepared by using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). The antigen–antibody complexes were detected with enhanced chemiluminescence. Antibodies used were anti-RECK (Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin (Sigma-Aldrich). β-Actin served as the internal control to eliminate the differences of protein loading.

2.6 Invasion assays

Invasion assays were performed with growth factor-reduced Matrigel®-coated filters (8 mm pore size; BD Biosciences, Franklin Lakes, NJ, USA) in 24 well plates. The transwells were coated with Matrigel in advance at 4 °C overnight, and then incubated at 37 °C for 1.5 h for solidification. After individual treatments, cells (2 × 10⁴) were suspended in FBS-free medium and seeded onto the upper chamber of Transwell plates. The lower chambers were filled with medium containing 100 ng mL⁻¹ of epidermal growth factor (R&D Systems, Minneapolis, MN, USA). The chambers were incubated at 37 °C for 24 h in the absence or presence of CO₂. At the end of the incubations, cells on the upper surface of the filter were removed gently with a cotton swab. Cells penetrating through the filter to the lower surface were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 10 min. Penetrated cells were photographed with a phase contrast microscope (Olympus, Tokyo, Japan) and counted in five randomly chosen fields.

2.7 Conditioned medium and gelatin zymography

Culture supernatants were prepared by incubating cells in respective medium containing 0.1% bovine serum albumin (BSA, volume: 300 mL per 5 × 10⁵ cells) at 37 °C for 12 h in the absence or presence of 5% CO₂. Proteins in the conditioned medium were separated, without prior boiling, by SDS-PAGE (10%) resolving gels containing 1 mg mL⁻¹ gelatin (Sigma-Aldrich) under non-reducing conditions. The proteins in the gel were renatured and stained with 0.05% Coomassie Brilliant Blue as described previously.²² Briefly, after electrophoresis, gels were washed twice in 2.5% Triton X-100/50 mM Tris–HCl, pH 7.5, and incubated within an incubator at 37 °C in the absence of CO₂ for 16 hours in renaturation solution contained 0.15 M NaCl/10 mM CaCl₂/50 mM Tris–HCI, pH 7.5/0.05% NaN₃. Then gels were stained with 0.005% Coomassie Blue R250 and destained with 10% (vol/vol) acetic acid and 10% (vol/vol) isopropanol. Type IV-collagenolytic enzymes were detected as transparent bands on slab gels.

2.8 Cell transfection

Anti-con, anti-miR-21, mimic-con, and mimic-miR-21 were purchased from RiBoBio, and control, and RECK small interfering RNA (siRNA) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were transiently transfected with Lipofectamine® 2000 reagent (Invitrogen) for 12 h following the manufacturer's instructions.

2.9 Statistical analysis

Values were presented as the mean ± SD. The one-way analysis of variance (ANOVA) followed by Dunnett's t-test were applied to determined statistical differences between groups. P-values < 0.05 were considered statistically significant.

3. Results

3.1 ISL attenuates the invasive capacity of breast cancer cells

We determined the effect of ISL on the viability of breast carcinoma cells with CCK-8 assays. Breast cancer cells MDA-MB-231 and Hs-578T were exposed to 0.0, 5.0, 10.0, 20.0, or 40.0 μM of ISL for 24 h and 48 h. ISL did not significantly change the viabilities of the breast cancer cells exposed to ISL at 0.0, 5.0, 10.0, and 20.0 μM for 24 h. In contrast, there were detectable decreases in viabilities of both cell lines treated with 40 μM of ISL following the 24 h incubation (Fig. 1A). Furthermore, ISL at 20 μM significantly reduced the viabilities of cells following the 48 h incubation (Fig. 1B). Therefore, 20 μM ISL for 24 h and 10 μM ISL for 48 h were selected as the highest concentrations for subsequent experiments. To determine the effects of ISL on the invasive capacity of breast cancer cells, invasion assays were performed. Both cell lines were treated with 0.0, 5.0, 10.0, or 20.0 μM of ISL for 24 h. As demonstrated in Fig. 1C and D, ISL decreased the invasive potential of breast cancer cells in a dose-dependent manner. These data indicated that ISL showed anti-tumor effects without affecting cellular viabilities.

Fig. 1 ISL attenuates the invasive capacity of breast cancer cells. Breast cancer cell lines MDA-MB-231 and Hs-578T cells were treated with ISL at 0.0, 5.0, 10.0, 20.0 and 40.0 μM for 24 h (A) or 48 h (B). The viability was measured using a cell counting kit-8 assay at 450 nm. The percentage of cell viability was calculated via comparing with non-treated cells (mean ± SD, n = 5). (C) Invasion assays were performed, and (D) invaded cell numbers were determined (mean ± SD, n = 5). **P < 0.01 compared with non-treated MDA-MB-231 cells and ##P < 0.01 compared with non-treated Hs-578T cells.

3.2 Effects of ISL on the matrix metalloproteinases (MMPs) in breast cancer cells

Since MMP2 and MMP9 acted as key mediators of the invasive process of cancer cells,^23,24 then we examined the effect of ISL on the expressions and activities of MMP2 and MMP9 in breast cancer cells. Cells were treated with ISL at 0.0, 5.0, 10.0 or 20.0 μM of ISL for 24 h, respectively. As shown in Fig. 2A, ISL could efficiently decrease the mRNA level of MMP9 in a dose-dependent manner in two breast cancer cell lines. Furthermore, MDA-MB-231 and Hs-578T cells treated with 0.0 or 10.0 μM ISL for different times up to 48 h, there were also decreased expressions of MMP9 in a time-dependent manner (Fig. 2B). However, no detectable effects were found on MMP2 expression in both cell lines demonstrated by dose- and time-course experiments (Fig. 2A and B). Subsequently, the gelatinase zymography analysis were performed to examine the activities of MMP2/9, and the results demonstrated that ISL attenuated the activities of MMP9 in both dose- and time-dependent manner. No detectable alterations of activities of MMP2 were presented (Fig. 2C and D). These results suggested that the inhibition of MMP9, not MMP2, was involved in the anti-tumor effect of ISL on breast cancer cells.

Fig. 2 Effects of ISL on the matrix metalloproteinases (MMPs) in breast cancer cells. MDA-MB-231 and Hs-578T cells were treated with ISL at 0.0, 5.0, 10.0 and 20.0 μM for 24 h. The mRNA (A) and enzyme activities (C) of MMP2 and MMP9 were investigated by RT-PCR and gelatin zymography assays, respectively. Breast cancer cell lines MDA-MB-231 and Hs-578T cells were treated with 10 μM ISL for 0, 12, 24 and 48 h. The mRNA (B) and enzyme activities (D) of MMP2 and MMP9 were determined by RT-PCR and gelatin zymography assays, respectively.

3.3 ISL increases RECK protein levels without affecting RECK mRNA levels in breast cancer cells

Puissant evidences showed that RECK could suppress the expression and activity of MMP9 via transcriptional repression and glycosylation, respectively.^25,26 As shown in Fig. 3A, the protein level of RECK increased in a dose-dependent manner as the breast cancer cells were exposed to ISL at different concentrations up to 20 μM for 24 h. As the time course experiments, RECK protein was also induced in a time-dependent manner by 10 μM of ISL in both cell lines (Fig. 3B). No detectable changes were observed for RECK mRNA levels in two cell lines treated with 0.0, 5.0, 10.0 and 20.0 μM of ISL for 24 h (Fig. 3C). Likewise, the RECK mRNA was unaffected in cells exposed to 10.0 μM of ISL for 0, 12, 24, and 48 h (Fig. 3D). The results suggested that RECK might be involved in the anti-tumor effects of ISL on breast cancer cells.

Fig. 3 ISL increases RECK protein levels without affecting RECK mRNA levels in breast cancer cells. MDA-MB-231 and Hs-578T cells were treated with ISL at 0.0, 5.0, 10.0 and 20.0 μM for 24 h. The reversion-inducing cysteine-rich protein with kazal motifs (RECK) protein (A) and mRNA (C) were investigated by western blots and quantitative real-time polymerase chain reaction (qRT-PCR) analysis (mean ± SD, n = 3), respectively. (B) Breast cancer cell lines MDA-MB-231 and Hs-578T cells were treated with 10 μM ISL for 0, 12, 24 and 48 h. RECK protein (B) and mRNA (D) were investigated by western blots and qRT-PCR analysis, respectively.

3.4 RECK induction is involved in the repressive effect of ISL on MMP9 and invasive potential of breast cancer cells

Herein, we used specific siRNA for RECK to determine the effects of RECK on the regulation of MMP9 in breast cancer cells. As shown in Fig. S1A† and 4A, the RECK siRNA could effectively knockdown the expression of RECK in the two highly invasive cell lines at mRNA and protein levels. To determine the role of induction of RECK in the inhibitory effect of ISL on MMP9, MDA-MB-231 and Hs-578T cells were transiently transfected with control or RECK siRNA for 12 h, then exposed to 0 or 10 μM of ISL for 24 h. The efficacy of transfection and effect of ISL on RECK expression were shown in Fig. S1B† and 4B. The MMP9 mRNA and enzyme activity, and the invasive potential of cells decreased or increased upon RECK up-regulation or down-regulation, respectively (Fig. 4C–E). Neither the mRNA nor the enzyme activity of MMP2 showed significant alteration upon RECK induction or reduction. These results above suggested that induction of RECK expression was involved in the anti-tumor effects of ISL on breast cancer cells.

Fig. 4 RECK induction is involved in the repressive effect of ISL on MMP9 and invasive potential of breast cancer cells. (A) The breast cancer cell lines MDA-MB-231 and Hs-578T were transiently transfected with control or RECK siRNA for 12 h. RECK protein were analyzed by Western blots analysis. The breast cancer cell lines MDA-MB-231 and Hs-578T were transiently transfected with control or RECK siRNA for 12 h, then exposed to 0.0 or 10.0 μM of ISL for 24 h. RECK protein (B) were analyzed by Western blots analysis. (C) RT-PCR analysis of MMP2 and MMP9 mRNA. (D) Gelatin zymography analysis of activity of MMP2 and MMP9. (E) Invasion assays were performed.

3.5 ISL up-regulates RECK protein level via the down-regulation of miR-21 expression in breast cancer cells

Next, we examined the mechanism whereby ISL induced RECK expression. Previous studies demonstrated that miR-21 could act as an oncogene via directly targeting the 3′-UTR within RECK mRNA.²⁷ We therefore determined the miR-21 expression status in cells exposed to ISL. As demonstrated in the dose and time course experiments, miR-21 was efficiently repressed by ISL in MDA-MB-231 and Hs-578T cells in both dose- and time-dependent manners (Fig. 5A and B).

Fig. 5 ISL up-regulates RECK protein level via the down-regulation of miR-21 expression in breast cancer cells. (A) MDA-MB-231 and Hs-578T cells were treated with ISL at 0.0, 5.0, 10.0 and 20.0 μM for 24 h. miR-21 expression was investigated by qRT-PCR analysis (mean ± SD, n = 3). (B) Breast cancer cell lines MDA-MB-231 and Hs-578T cells were treated with 10 μM ISL for 0, 12, 24 and 48 h. miR-21 expression was investigated by qRT-PCR analysis (mean ± SD, n = 3). Breast cancer cell lines Hs-578T and MDA-MB-231 cells were transiently transfected with anti-con or anti-miR-21 for 12 h. RECK protein (C) and mRNA (D) were examined by Western blots and qRT-PCR (mean ± SD, n = 3), respectively. The breast cancer cell lines MDA-MB-231 and Hs-578T were transiently transfected with mimic-con or mimic-miR-21 for 12 h, then exposed to 0.0 or 10.0 μM of ISL for 24 h. RECK protein (E) and mRNA (F) were investigated by Western blots and qRT-PCR (mean ± SD, n = 3), respectively. *P < 0.05 and **P < 0.01 compared with non-treated MDA-MB-231 cells, respectively; #P < 0.05 and ##P < 0.01 compared with non-treated Hs-578T cells, respectively.

Breast cancer cells were then transfected with anti-con and anti-miR-21 for 12 h. The expression of miR-21 decreased upon anti-miR-21 transfection, indicating that the transfections were effective in both cell lines (Fig. S1C†). As expected, the RECK protein levels increased with successful miR-21 down-regulation in cancer cells (Fig. 5C), suggesting that RECK was negatively regulated by miR-21 in MDA-MB-231 and Hs-578T cells. However, no significant differences were observed on the mRNA level of RECK between two groups in both cell lines (Fig. 5D). These results demonstrated that miR-21 inhibited RECK protein expression via translation repression rather than mRNA degradation induction in breast cancer cells. Subsequently, MDA-MB-231 and Hs-578T cells were transiently transfected with mimic-con or mimic-miR-21 for 12 h, then exposed to 10 μM of ISL for 24 h. The efficiency of transfection is shown in Fig. S1D.† As presented here, ectopic miR-21 expression by miR-21 mimics could reverse the inductive effect of ISL on RECK protein levels in breast cancer cells without changes of RECK mRNA levels (Fig. 5E and F).

3.6 ISL reduces the invasive potential of breast cancer cells via decreasing miR-21

As demonstrated in Fig. 6, ISL attenuated the invasive capacity of breast cancer cells, as determined by invasion assays; however, restoration of miR-21 abrogated such effects. These results suggested that inhibition of miR-21 was involved in the anti-tumor effects of ISL breast cancer cells.

Fig. 6 ISL reduces the invasive potential of breast cancer cells via decreasing miR-21. MDA-MB-231 and Hs-578T cells were transiently transfected with mimic-con or mimic-miR-21 for 12 h, then exposed to 10.0 μM of ISL for 24 h. (A) and (C) Invasion assays were performed. (B) and (D) Invaded cell numbers were determined. **P < 0.01 compared with mimic-con transfected cells and ##P < 0.01 compared with mimic-con-transfected cells treated by 10 μM of ISL.

4. Discussion

Despite advances in early diagnosis system and multidisciplinary treatment, breast cancer remains the most commonly diagnosed malignancy among women worldwide.¹ Moreover, enormous data revealed that metastasis was the major cause of the deaths of breast cancer patients due to complicated process and less effective treatments.²

Enormous evidence supported the idea that breaking through the extracellular matrix mediated by the MMPs, a key event in tumor cell invasion, played a vital role in tumor metastasis.^28,29 The MMPs, consisted of more than 20 members, are a class of Zn²⁺-dependent endopeptidases with the ability to degrade extracellular matrix proteins resulting in matrix remodeling and cancer cellular invasion.³⁰ Actually, the MMPs had been regarded as breast cancer drivers and therapeutic targets.³¹ Nowadays, some inhibitors for MMPs have been tested in clinical trials, however, some side effects appeared, mainly due to the limited selectivity of these inhibitors.³² Among the MMPs, MMP2 and MMP9 were considered as critical mediators of tumor metastasis due to their connatural capacity of degrading type IV collagen, a major component of extracellular matrix.^23,24 Furthermore, the deregulation of MMP2 and MMP9 had been found during breast cancer metastasis and negatively correlated with survival of breast cancer patients.^33,34 In our study, we revealed that ISL, extracted from liquorice, showed anti-tumor effects via reducing the expression and activity of MMP9, but not MMP2, in breast cancer cells in both dose- and time-dependent manners, accompanied by reduction of invasiveness of breast cancer cells (Fig. 1 and 2).

The RECK gene has been investigated for almost twenty years as a tumor suppressor for its repressive activity on MMP9 and MMP2.^3,7,25,26 Moreover, in enormous clinical samples, researchers also revealed that the RECK-status is a significant prognostic factor for breast cancer, namely down-regulated RECK expression correlated with poor prognosis.³⁵ RECK down-regulation was frequently found in many types of cancer-derived cell lines.³ The results of a number of published reports indicated that the SP1 site located in the proximal 52bp from transcription start site of the RECK promoter, histone deacetylase 1 (HDAC1) and DNA methyltransferase 3b (DNMT3b) were participated in the repression of RECK by oncogenes.^4–6 As demonstrated here, ISL could significantly up-regulate RECK expression in cancer cells and induction of RECK was proved the involvement in the anti-tumor effect of ISL on breast cancer cell (Fig. 4). The results of a previous study had shown the inhibitory effect of ISL on cancer cellular invasiveness. Indeed, inactivated PI3K/Akt pathway, the phosphorylation of p38 and NF-κB DNA binding activity-induced inhibition of MMP2 and MMP9 may be responsible for the repressive effect of ISL on the migration and invasion of breast cancer cell MDA-MB-231.²¹ As presented in our research, inhibition of MMP9, but not MMP2, mediated the anti-tumor activity of ISL, which may be due to different glycosylation sites in RECK protein. Previous evidence showed that RECK exerted repressive effect on MMP9 and MMP2 via glycosylation of at the sites of Asn297 and Asn352, respectively.²⁶ In our study, we did not observe any alterations of MMP2 upon the induction of RECK due to treatment of ISL. This may result from preserved glycosylation of Asn297 but absent Asn352 glycosylation in breast cancer cells. A former research on RECK in colorectal cancer revealed an inverse relation between RECK and MMP2 but not with MMP9. The researcher speculated that it may be due to preserved glycosylation of Asn352 but absent Asn297 glycosylation in colorectal carcinoma.³⁶

The microRNAs are a class of small noncoding RNAs, binding to the 3′-UTR of target mRNA and resulting in target mRNA translation repression or degradation, regulates the expression of specific protein-coding mRNA at post-transcriptional level.⁸ Mounting evidence revealed the involvement of miRNAs in many cancer cellular processes, such as cellular invasion.¹¹ For example, overexpression of miR-21 has been well documented in many types of human cancer, including prostate, breast, lung, colon, stomach and pancreatic tumors.³⁷ Furthermore, highly expressed miR-21 correlated positively with cancer cell invasion. It has already been demonstrated that miR-21 may promote tumor progression by targeting PDCD4 and PTEN, two important tumor suppressors.³⁸ Moreover, miR-21 could also directly bind to specific sites within the 3′-UTR of RECK mRNA and negatively regulate its expression, which has been widely proved in kinds of cancer-derived cell lines, such as human fibrosarcoma and colon carcinoma cells.²⁷ To our knowledge, no reports revealed whether ISL could reduce the expression of miR-21 in cancer cells. In our study, we found that ISL could efficiently increase the protein level of RECK without affecting its mRNA level. Further mechanism investigations revealed that repression of miR-21 was involved in the inductive effect of ISL on RECK expression. Moreover, we firstly found that miR-21 negatively regulate RECK expression via translation repression rather than mRNA degradation in breast cancer cells.

In the present study, we did not elucidate the mechanism whereby ISL reduced miR-21 expression. Previous studies showed that miR-21 could be transcriptionally controlled by lots of transcriptional factors, including AP-1, NF-κB, STAT3 and ETS-1.^39–42 Perhaps, more than a single transcriptional factor mediates the repressive effect of ISL on miR-21 expression.

5. Conclusion

Taken together, our study showed decreased miR-21 expression was found in MDA-MB-231 and Hs-578T breast cancer cells exposed to ISL. ISL treatment increased RECK protein, but not RECK mRNA, and this was accompanied by a reduction of cellular invasiveness. By identifying a novel mechanism by which ISL, a phytochemical widely existed in our dietary supplement, negatively regulate breast cancer cell invasion via induction of RECK, our study not only expand understanding of the anti-tumor potential of ISL, but also provide an alternative for the treatment of breast cancer.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81171987), a Research Fund for the Doctoral Program of Higher Education of China (20133234110007), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra26759e

‡ Authors contributed equally to this work.

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