Adenosine dialdehyde suppresses MMP-9-mediated invasion of cancer cells by blocking the Ras/Raf-1/ERK/AP-1 signaling pathway

Abstract

Adenosine dialdehyde (AdOx) inhibits transmethylation by the accumulation of S-adenosylhomocysteine (SAH), a negative feedback inhibitor of methylation, through the suppression of SAH hydrolase (SAHH). In this study, we aimed to determine the regulatory effect of AdOx on cancer invasion by using three different cell lines: MDA-MB-231, MCF-7, and U87. The invasive capacity of these cells in the presence (MCF-7) or absence (MDA-MB-231 and U87) of phorbal 12-myristate 13-acetate (PMA) was strongly decreased by AdOx treatment. Furthermore, the expression, secretion, and activation of matrix metalloproteinase (MMP)-9, a critical enzyme regulating cell invasion, in these cells were diminished by AdOx treatment. AdOx strongly suppressed AP-1-mediated luciferase activity and, in parallel, reduced the translocation of c-Fos and c-Jun into the nucleus. AdOx was shown to block a series of upstream AP-1 activation signaling complexes composed of extracellular signal-related kinase (ERK), mitogen-activated protein ERK kinase (MEK)1/2, Raf-1, and Ras, as assessed by measuring the levels of the phosphorylated and membrane-translocated forms. Furthermore, we found that suppression of SAHH by siRNA and 3-deazaadenosine, knock down of isoprenylcysteine carboxyl methyltransferase (ICMT), and treatment with SAH showed inhibitory patterns similar to those of AdOx. Therefore, our data suggest that AdOx is capable of targeting the methylation reaction regulated by SAHH and ICMT and subsequently downregulating MMP-9 expression and decreasing invasion of cancer cells through inhibition of the Ras/Raf-1/ERK/AP-1 pathway.

Introduction

It is well-known that when a tumor metastasizes [1], the cancer becomes very difficult to cure. Six steps lead to metastasis: (1) aggressive proliferation of transformed cells; (2) breakdown of the basal lamina; (3) intravasation and circulation through the bloodstream or lymphatic system; (4) attachment to the inner wall of the blood vessel; (5) extravasation; and (6) abnormal proliferation at the metastasized site [2], [3]. Among these steps, acquisition of invasive capacity is a key step toward cancer malignancy.

Tumor cell invasion, i.e., the expansion of tumor cells into surrounding tissues, is a complex process that leads to proteolysis and destruction of biological barriers [4]. Type I collagen is the most abundant component of the extracellular matrix, and the invasive capacity of tumor cells involves proteolytic enzymes such as matrix metalloproteinases (MMPs) [5]. Among the extracellular proteases responsible for the degradation of the extracellular matrix, MMP-2 and MMP-9 play critical roles in the invasiveness of carcinoma [6], [7]. Therefore, targeting of the enzyme activity of MMP has been regarded as an important strategy in the development of anti-tumor agents with anti-invasive properties.

Methylation is a representative biochemical reaction in which a methyl group from S-adenosylmethionine (SAM) is transferred to numerous methyl-accepting donors such as proteins, DNA, and RNA [8], [9]. This reaction is catalyzed by various protein/DNA/RNA methyltransferases (N-, O-, and S-methyltransferases) [10], [11]. Although methylation is regarded as a critical pathway in many different human diseases including aging, obesity, and Parkinson's disease, it is unclear how methylation modulates tumorigenic responses [12]. In particular, because epigenetic processes such as DNA methylation and histone modifications, including methylation and acetylation, at lysine and arginine residues are tightly associated with the proliferation, apoptosis, survival, migration, and invasion of tumor cells [13], it is expected that elucidation of the methylation reaction could lead to therapeutic solutions.

Adenosine dialdehyde (AdOx) is a broadly used indirect methylation inhibitor [10], [14], [15]. AdOx inhibits adenosylhomocysteine hydrolase (SAHH), which is an important enzyme for SAM metabolism [16], [17]. SAHH converts S-adenosyl-l-homocysteine (SAH), a negative feedback inhibitor of methylatransferase that utilizes S-adenosyl-l-methionine (SAM) as its methyl donor, into adenosine and l-homocysteine, which are necessary for producing SAM [18]. AdOx treatment induces a hypomethylated state by suppressing methyltransferase activity in cells in two ways: (1) accumulation of SAH and (2) deficiency of SAM [19], [20]. Whereas the functional role of AdOx in the methylation of tumorigenic responses is known, the inhibitory activity of AdOx in various cancer stages has not been fully elucidated. Indeed, this compound has been shown to block the proliferation of some cancer cells such as prostate cancer cells, P19 embryonic carcinoma cells, MCF breast cancer cells, and neuroblastoma cells [10], [21], [22]. In particular, AdOx is able to arrest the cell cycle at the G₂ phase and induce p53-dependent apoptosis [11], [23]. These results strongly suggest the possibility that AdOx can be used as a methylation-inhibitory anti-cancer drug. However, no studies have demonstrated the suppressive role of AdOx in tumor cell invasion and migration. The aim of this study, therefore, was to evaluate the inhibitory effect of AdOx on tumor invasion in view of its molecular suppressive mechanism.