GM6001

Inhibition of MMP-2-mediated cellular invasion by NF-B inhibitor DHMEQ in 3D culture of breast carcinoma MDA-MB-231 cells: A model for early phase of metastasis

Tamami Ukajia, Yinzhi Lina, Shoshiro Okadab, Kazuo Umezawaa, *

Abstract

The three-dimensional (3D) culture of cancer cells provides an environmental condition closely related to the condition in vivo. It would especially be an ideal model for the early phase of metastasis, including the detachment and invasion of cancer cells from the primary tumor. In one hand, dehydroxymethylepoxyquinomicin (DHMEQ), an NF-B inhibitor, is known to inhibit cancer progression and late phase metastasis in animal experiments. In the present research, we studied the inhibitory activity on the 3D invasion of breast carcinoma cells. Breast carcinoma MDA-MB-231 cells showed the most active invasion from spheroid among the cell lines tested. DHMEQ inhibited the 3D invasion of cells at the 3D-nontoxic concentrations. The PCR array analysis using RNA isolated from the 3D on-top cultured cells indicated that MMP-2 expression is lowered by DHMEQ. Knockdown of matrix metalloproteinase (MMP)-2 and an MMP inhibitor, GM6001, both inhibited the invasion. DHMEQ was shown to inhibit the promoter activity of MMP-2 in the reporter assay. Thus, DHMEQ was shown to inhibit NF-B/MMP-2-dependent cellular invasion in 3D-cultured MDA-MB-231 cells, suggesting that DHMEQ would inhibit the early phase of metastasis.

Keywords
3D culture; invasion; MDA-MB-231 cells; DHMEQ; MMP-2;

Introduction

Metastasis inhibitors without cellular toxicity should be useful for anticancer agents with few side effects. Until recently, the cell-based study of cancer growth and metastasis has been carried out mainly in monolayer 2D cultured cells, as with the wound healing assay and Matrix chamber assay [1]. However, cancer cells grow and metastasize in the body with the 3D organization interacting with neighboring cancer cells. Recently, invasion of cancer cells has become available for study in the 3D condition [2]. In the 3D invasion assay, firstly, multicellular spheroids are prepared. Then, they are embedded in the extracellular matrix, such as the basement membrane extract (BME) or collagen I [3].
The process of metastasis includes detachment of the cells from the primary tumor, migration and invasion, transportation to a remote place through the vessels, attachment and growth of the secondary tumor. There exist many in vivo models of the later stages, such as attachment of cells and formation of secondary tumor. However, earlier metastasis models which study the detachment from the primary tumor and invasion are very rare. The 3D culture of cancer cells should be an ideal model of these earlier processes of metastasis (Fig. 1).
On one hand, we previously discovered DHMEQ (Fig. 2A) by molecular design as an inhibitor of NF-B [4]. It binds to the specific cysteine residues of NF-B components, including p65, RelB, cRel, and p50, to inhibit their DNA binding [5,6]. It was shown to ameliorate various animal models of cancer and inflammation without showing any side effect [7]. Previously, it was shown to inhibit Matrigel invasion in cultured ovarian clear cell carcinoma RMG1 cells [8]. Intraperitoneal administration of DHMEQ inhibited liver metastasis of pancreatic carcinoma AsPC-1 cells injected via the portal vein [9]. However, the effect of DHMEQ on the earlier metastatic process, on cell detachment from the primary tumor and on invasion, has never been tested.
In the present research we studied the effect of DHMEQ on the invasion of 3D-cultured cancer cells. DHMEQ inhibited the 3D-invasion of breast carcinoma MDA-MB-231 cells without toxicity, by inhibition of MMP-2 expressions.

Materials and Methods

Materials

DHMEQ was synthesized in our laboratory, as previously described [10]. MMP inhibitor GM6001 was purchased from Abcam (Cambridge, United Kingdom). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich (St. Louis, MO). The ELISA kit for IL-6 was purchased from R&D Systems, Minneapolis, MN.

Cell culture

Human breast cancer MDA-MB-231 cells were purchased from RIKEN Cell Bank (Tsukuba, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) fetal bovine serum and penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2.

Cell viability assays

Cell viability was evaluated by MTT assay performed as previously reported [1].

NF-B binding assay

NF-B binding activity was measured as previously reported [11] with slight modifications. The 2D-cultured cells were grown in 100 mm dishes. The following day, the cells were treated with DHMEQ for 2 h.

Wound healing assay and cell invasion assay

Wound healing and Matrigel cell invasion assays were performed as previously reported [1].

RNA isolation and semi-quantitative RT-PCR analysis

These assays were performed as previously reported [1]. The primers used for semi-quantitative RT-PCR, the number of cycles, and the annealing temperature, as follows: MMP-2, 5’- AAG TCT GGA GCG ATG TGA CC -3’ (forward) and 5’- GAGTCC GTC CTT ACC GTC AA -3’ (reverse), 34 cycles, 58°C; and -actin, 5’- CTT CTA CAA TGA GCT GCG TG-3’ (forward) and 5’- TCA TGA GGT AGT CAG TCAGG-3’ (reverse), 21 cycles, 58°C. PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide, and visualized with a UV illuminator.

3D spheroid proliferation assay

The 3D spheroid proliferation assay kit was purchased from Trevigen Inc. (Gaithersburg, MD) and used according to the manufacturer’s protocol. After incubation with chemical for several days, proliferation was assessed by MTT.

3D spheroid cell invasion assay

3D spheroid cell invasion assay kit was purchased from Trevigen Inc. and used according to the manufacturer’s protocol. For analysis, spheroids were photographed in each well every 24 h. Spheroid Area were measured using Image J (National Institute of Health).

On-top 3D cell culture and harvest

The 3D on-top method was followed as previously reported [12]. 3D culture Matrix Reduced Growth Factor Basement Membrane Extract (BME) was purchased from Trevigen Inc. Cells were cultured for 4 days and the medium was changed every 2 days. Then the medium was replaced to the new one including DHMEQ and incubation was for 24 h. Cell supernatants were used for the ELISA assay. The 3D culture matrix cell harvesting kit (Trevigen Inc.) was used according to the manufacturer’s protocol, and harvested cell lysates were used for array analysis and RT-PCR.

PCR array

Total RNA was extracted from 3D-cultured MDA-MB-231 cells using RNeasy mini (Qiagen, Hilden, Germany). The cDNA was added to the qPCR Master Mix and the aliquot mixture across the Human Tumor Metastasis PCR Array (Qiagen). Data analysis was carried out by the comparative Ct method.

Knockdown of MMP-2 by siRNA treatment

siMMP-2 for human (sc-29398) and small interfering RNA-A as control (sc-37007) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) Transfection of cells with siRNAs was carried out as previously reported [1]. The efficiency of transfection for 96 hours was determined by mRNA expression.

Plasmid construction

The DNA fragment (bp -1259 to +57) upstream from the transcription initiation site of human MMP-2 was amplified by PCR with KOD plus DNA polymerase (Toyobo). A human MMP-2 promoter was a gift from Dr. Etty Benveniste (Addgene plasmid # 53432) and used as the template [13]. The pGL3-Basic vector (Promega, Madison, WI) was used to construct expression vectors by subcloning PCR-amplified DNA of human MMP-2 promoter into the XhoI/HindIII site of the pGL3-Basic vector. Each clone was confirmed by DNA sequencing.

DNA transfection and luciferase assay

MDA-MB-231 cells were seeded at a density of 4 x 105/6-well plate and transiently transfected with pGL3-basic or pGL3-MMP-2 promoter vector and pRL-TK vector (2g and 100 ng, respectively) following the standard Lipofectamine LTX (Life Technologies, Carlsbad, CA) transfection protocols. Reporter assays for promoter activity were carried out using the Dual-Glo Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase in each well.

Statistical analysis

All experiments were performed independently at least three times. Differences between groups were examined for significance with ANOVA and/or the Student’s t-test where appropriate.

Results

Selection of cell line and time-course of 3D invasion

We evaluated 5 highly malignant cell lines including breast carcinoma human MDA-MB-231, human fibrosarcoma HT-1080, human pancreatic carcinoma AsPC-1 [9], human ovarian clear cell carcinoma ES-2 [1], and mouse melanoma B16-F10 [14] cells. Among them, ES-2, AsPC-1, and B16-F10 cells showed no invasion, and HT-1080 cells weakly showed the invasion. MDA-MB-231 shows 3D detachment/invasion clearly, and we mainly employed MDA-MB-231 cells for further experimentation. Fig. 1 shows the time-course of 3D invasion in MDA-MB-231 cells.

Inhibition of invasion and NF-B in 2D-cultured MDA-MB-231 cells.

DHMEQ (Fig. 2A) is not toxic below 10 g/ml (Fig. 2B) in 2D-cultured MDA-MB-231 cells. It inhibited invasion in the Matrigel chamber at 1-10 g/ml (Fig. 2C). MDA-MB-231 cells possess constitutively activated NF-B [14], which was inhibited by DHMEQ (Fig. 2D). IL-6 is dependent on NF-B, and its secretion was also inhibited by DHMEQ (Fig. 2E).

Inhibition of invasion and MMP-2 expression in 3D-cultured MDA-MB-231 cells.

Firstly, we measured the toxicity of DHMEQ in 3D-cultured cells. As shown in the illustration in Fig. 3A, DHMEQ was added after spheroid formation. DHMEQ did not lower the viability even at 50 g/ml (Fig. 3A). Next, DHMEQ was shown to inhibit 3D-invasion at 10-30 g/ml, as shown in Fig. 3B.
Next, we studied the mechanism of inhibition by DHMEQ employing a PCR array analysis targeting tumor metastasis. As shown in the illustration in Fig. 3C, we employed the BME gel and cultured cells in the 3D on-top method [12]; then, DHMEQ was added for 24 h. Only a few gene expressions were found to be lowered in the array analysis. Among them, DHMEQ inhibited the expression of MMP-2 about 60%. Then, we confirmed the decrease independently by PCR employing the mRNA from the 3D-cultured cells. As a result, DHMEQ inhibited the MMP-2 expression (Fig. 3D). Since the amount of nuclei from the 3D-cultured cells was not enough for NF-B analysis, we measured the downstream IL-6 secretion. DHMEQ lowered the secretion of IL-6 (Fig. 3E).

Inhibition of 3D-invasion by MMP-2 siRNA or MMP inhibitor.

To confirm that MMP-2 is the functional target for the inhibition of invasion, we have knocked down MMP-2 by siRNA. Expression of MMP-2 was suppressed even after the formation of the spheroid (Fig. 4A). Knockdown of MMP-2 did not affect 3D cell proliferation (Fig. 4B), but the 3D invasion was inhibited by knockdown of MMP-2 (Fig. 4C). Furthermore, DHMEQ did not lower the 3D invasion in siMMP-2-treated cells (Fig. 4D). GM6001 is an inhibitor of several MMPs, including MMP-2. It showed no toxicity even at 30 M (Fig. 4E), and also inhibited 3D invasion at 3-10 M (Fig. 4F). Moreover, DHMEQ did not further lower the 3D invasion in GM6001-treated cells, as shown in Fig. 4G. Thus, DHMEQ inhibited 3D invasion by down-regulation of MMP-2 expression via NF-B inhibition.
Finally, to show the inhibition of promoter activity by DHMEQ, we constructed the reporter plasmid containing the MMP-2 promoter sequence and luciferase gene. Using this plasmid DHMEQ was shown to inhibit the promoter activity, as shown in Fig. 4H.

Discussion

These 3D culture systems are useful for the mechanistic study of the early steps in metastasis and for the screening of metastasis inhibitors. Fig. 1 clearly shows relation of the 3D invasion experiment and early phase of metastasis, detachment and invasion. DHMEQ inhibited this early stage of metastasis. Moreover, it also inhibited the 3D invasion of HT-1080 cells (data not shown).
NF-κB is often constitutively activated in cancer cells [15] including human breast cancer MDA-MB-231 cells [16]. NF-κB of tumor cells should activate the detachment and invasion via the activation of MMP-2 from the primary tumor. Secretion of MMP-2 by tumor cells degrades the tissue matrix to facilitate invasion. Moreover, the cell surface proteins that are essential for the cell-cell attachment should be degraded by MMP-2 to facilitate the detachment of tumor cells.
We demonstrated that DHMEQ inhibited the MMP-2 promoter activity using the reporter plasmid (Fig. 4H). Epanchintsev et al. also reported that NF-κB inhibitor BAY 11-7082 inhibited the promoter activity of MMP-2, as well as migration, in MDA-MB-231 cells [17]. Thus, DHMEQ is likely to inhibit NF-κB dependent MMP-2 expression via direct transcript inhibitory activity [18]. DHMEQ has been reported to inhibit various disease models in animal experiments thus far without any toxicity. It may be a non-toxic anti-metastasis agent acting both at the early and late phases of metastasis.

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