Original article
RY10-4 suppressed metastasis of MDA-MB-231 by stabilizing ECM and E-cadherin

https://doi.org/10.1016/j.biopha.2014.03.003Get rights and content

Abstract

In the article, we investigated the anti-metastasis mechanism of RY10-4, an anti-tumor compound derived from protoapigenone, in breast tumor cells MB-MDA-231. The analog of protoapigenone with an unaromatic B-ring was verified to suppress the proliferation of several tumor cells by previous research that also showed that several tumor progression such as inducing apoptosis and anti-angiogenesis could be acted on by RY10-4. In the article, we investigated the mechanism about how RY10-4 suppressed the invasion of MDA-MB-231. Firstly, the transwells assays with and without matrigel were adapted to evaluate the anti-metastasis and anti-invasion activity. Much research had demonstrated that the ECM and E-cadherin/β-catenin complex play an important role in cell adhesion and the formation of the cell skeleton, and as we knew the abnormal and absent expression of ECM and E-cadherin/β-catenin complex are found in many malignant cells. The result demonstrated that the amount and distribution of E-cadherin/β-catenin complex were backed on track by RY10-4, and the expression of MMP-2/9 in MDA-MB-231, which functions as a major negative factor of ECM, was down-regulated after co-cultured with RY10-4. Furthermore the pathway related to MMP-2/9 and E-cadherin was assessed by the western blot. As the results showed, the MAPK pathway and the spread of β-catenin were affected by RY10-4 to exert the anti-metastasis on MDA-MB-231. Collectively, the research revealed a novel anti-tumor ability of RY10-4 by inhibiting migration and invasion in MDA-MB-231.

Introduction

Human breast metastatic cancer is the second largest cause of cancer-related deaths among women in western countries [1]. Tumor metastasis is a complex process containing several steps in which cancer cells become mobile. Several main events happen in tumor metastasis that mainly involve the degradation of the extracellular matrix (ECM), entering the blood and lymphatic vessels, attaching to a new tissue [2]. Metastatic cells should then survive to anoikis, despite not having a proper ECM attachment, elude immunosurveillance, home in on target distant sites, and finally grow in the new environment [3]. The subtypes of breast cancer are generally divided according to the presence or absence of three receptors: estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER-2). MDA-MB-231 was regarded prone to migrate to other tissue among the breast cancer cells [4]. Thus MDA-MB-231 became the target cancer cells in anti-metastatic research.

ECM can be regulated by both positive and negative factors, as TIMPs and MMPs, both of which can be adjusted by many pathways such as MAPK [5]. Degradation of extracellular matrix (ECM) is a hallmark for invasion and metastasis of tumor cells. Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, play a considerable role in several aspects such as remodeling of extracellular matrix structures in wound tissue and in several pathologies such as cancerogenesis [6]. Specifically, MMP-2/9 were reported to frequently over-express in malignant tumors and the activity of MMP-2/9 dominated the degradation of the collagen type IV, the primary component of basement membranes. In all, MMPs may account for the normal pathological progression and the development of tumor [7].

Catenins are a kind of cytoplasmic proteins that dominated the interaction of the intracellular protein in epithelial cells [8]. The main structure of the cadherin complex is composed of the following components: cadherin, intracellular components associated with cadherin, and the cytoskeleton. The cadherin found in epithelial cells is named as E-cadherin [9]. The association between loss or down-regulation of E-cadherin and the progression of breast cancer has been extensively reported. The loss of E-cadherin mediated cell adhesion is a hallmark of tumor cells metastasis [10]. β-catenin is the transducer/transcriptional regulator of the Wnt signaling pathway, and also functions as a connection with E-cadherin cytoplasmic domain. As we know, the Wnt signaling pathway is involved in many developmental processes and tumorigenesis. The pathway is usually regulated by accumulating of β-catenin in the nucleus, where it interacts with TCF/LEF transcription factors, leading to activation of downstream target genes [11]. Before the translocation of β-catenin into nucleus, β-catenin can be phosphorylated and degraded in cytoplasm. So both the amount and the distribution of β-catenin not only affect the tumor progression by regulating Wnt pathway, but also determine the intactness of E-cadherin/β-catenin complex [12], which prevent the tumor cells from migration and invasion.

Heteroaromatic quinols with the structure of 4-hydroxycyclohexa-2,5-dienone exhibited potent anti-tumor activity [13]. RY10-4, a novel anti-tumor compound derived from protoapigenone with the structure as Fig. 1 showed, has been proved to induce apoptosis and inhibit angiogenesis [14]. In the study, we investigated the anti-metastasis effect and mechanism of RY10-4 in MDA-MB-231 cells, which are highly metastatic human breast cancer cells. In this paper, we verified the activity of RY10-4 to suppress tumor cells metastasis by stabilizing ECM and stimulating the expression of E-cadherin. Furthermore we evaluated the expression of MMP-2/9 and the key proteins in MMP-2/9 related pathway MAPK by the western blot. The result of the western blot showed that RY10-4 prevent the ECM from degradation in MDA-MB-231 by down-regulating MMP-2/9, and the activity of upstream signal pathway MAPK were weakened by down-regulating the phosphorylation levels of ERK1/2, P38, JNK1/2, furthermore the activation of upstream protein c-Scr and FAK were blocked by RY10-4.

Section snippets

Cell lines, chemical and isolation of nuclear and cytosolic fractions

MDA-MB-231 was obtained from Tongji Hospital, Wuhan, China, and cultured in RPMI 1640 medium supplemented with 20% FBS and 100 U/mL penicillin-streptomycin in an atmosphere of 5% CO2 at 37 °C in a humidified incubator. Nuclear and cytosolic fractions were isolated from cells according to the manual for NE-PER Nuclear and Cytoplasmic Extraction Reagents Kits (Thermo Scientific) [11]. Compound 1-hydroxycyclohexa-2, 5-dien-4-one group (RY10-4) was synthesized by our lab according to the previous

Suppressing the adhesion of MDA-MB-231 cell to fibronectin

Attaching to the new environment was crucial to tumor cells metastasis [17]. When MDA-MB-231 co-cultured with RY10-4, the number of cells adhered to the fibronectin decreased. As seen in Fig. 2, RY10-4 prevents the MDA-MB-231 from attaching to the fibronectin. The percentage rate of MDA-MB-231 attaching to fibronectin was about 75% and 32% with 0.5 and 2.5 μM RY10-4 respectively, compared to the vehicle control.

Inhibition of cells migration and invasion

The migration and invasion assays differ from the absence and presence of matrigel.

Discussion

In the research, RY10-4, of which the anti-tumor activity has been verified by previous study, was demonstrated to stabilize ECM and E-cadherin to prevent MDA-MB-231 cells from metastasis. MDA-MB-231 is a kind of breast cell that was regarded to prone to metastasis. Aberrant motive and metastasis ability of cancer cells are regarded as the important biological characteristics of cancers. The ability of cells to become locally invasive connected with the loss of E-cadherin-mediated cell to cell

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgments

This research was gratefully acknowledged by a grant (No. 30973864) from the National Natural Science Foundation of China and a grant (No. 2009CDA067) from the Nature Science Foundation of Hubei Province.

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