Oroxylin A inhibits matrix metalloproteinase-2/9 expression and activation by up-regulating tissue inhibitor of metalloproteinase-2 and suppressing the ERK1/2 signaling pathway

Abstract

Matrix metalloproteinases (MMPs) play important roles in the invasion and migration of cancer cells. In this study, we used in vitro and in vivo assays to examine the inhibitory effects of oroxylin A, one of the main bioactive flavonoid extracted from Scutellaria radix, on the human breast carcinoma cell MDA-MB-231 invasion and migration. We found that oroxylin A can suppress cell adhesion, invasion and migration in a concentration-dependent manner. Moreover, oroxylin A led to the reduction of the activity and expression levels of MMP-2 and MMP-9 in gelatin zymography, real-time PCR and western blotting analysis. Further elucidation of the mechanism revealed that oroxylin A increased the expression of tissue inhibitor of metalloproteinase-2 (TIMP-2), the endogenous inhibitor of MMP-2, and repressed the phorbol-12-myristate-13-acetate (PMA)-induced translocation of protein kinase Cδ (PKCδ), phosphorylation of extracellular signal-regulated kinase (ERK1/2) and binding activity of the transcription factor activator protein-1 (AP-1) which are upstream signaling molecules in MMP-9 expression. Our results also indicated that oroxylin A inhibited the lung metastasis of murine melanoma cell B16-F10 in vivo. Therefore, we proposed that oroxylin A might be developed as a therapeutic potential candidate for the treatment of cancer metastasis.

Introduction

Cancer metastasis is composed of a complex series of phenotypic and biochemical changes such as angiogenesis, lymphangiogenesis, gene expression, motility or cell shape (Steeg and Theodorescu, 2008). Excess matrix degradation is one of the hallmarks of cancer metastasis and is an important component of the process of tumor progression. Many proteinases play important roles in tumor metastasis, but one family of enzymes that appears to be particularly pivotal for matrix degradation is the matrix metalloproteinases (MMPs). MMPs are the principal enzymes in extracellular matrix (ECM) degradation which is essential in the invasive growth and metastasis of cancer (McCawley and Matrisian, 2000).

MMPs are structurally and functionally homologous proteins with clear links to malignancy. Tumor-secreted MMPs destroy ECM components in the surrounding tissues of the tumor, and subsequently tumor cells invade through the basement membrane of blood vessels and facilitate the spread to distant organs, resulting in organ failure and patient mortality (Nelson et al., 2000). More than twenty enzymes are classified as MMPs which also can be divided into collagenases, gelatinases, stromelysins and elastases by the substrate-based classification. Among the MMPs, MMP-2 (gelatinase A, 72 kDa) and MMP-9 (gelatinase B, 92 kDa) are gelatinases, which are considered to play vital roles in tumor invasion and metastasis. These two gelatinases share structural and catalytic similarities, but transcription of the MMP-2 and MMP-9 genes is independently regulated due to distinct arrays of regulatory elements in the gene promoters. Both of these MMPs are abundantly expressed in various malignant tumors (John and Tuszynski, 2001, Mook et al., 2004). Therefore, MMP-2 and MMP-9 are prime candidates for cancer therapy because they can collectively degrade all structural components of the ECM. Moreover, MMP-2 and MMP-9 are up-regulated in virtually all human and animal tumors and appear to increase with developing stages of tumor progression (Coussens et al., 2002).

Oroxylin A (Fig. 1A) is one of the main bioactive flavonoids extracted from Scutellaria radix, the root of Scutellaria baicalensis, a conventional herbal medicine widely prescribed as an analgesic, antipyretic, anti-inflammation, anticancer, antiviral and antibacterial infections remedy (Tayarani-Najaran et al., 2010). Recently, the antitumor activity of oroxylin A has attracted much attention. The previous studies have demonstrated that oroxylin A could induce apoptosis of human hepatocellular carcinoma cell HepG2 and human cervical cancer Hela cell in vitro and in vivo (Hu et al., 2006, Li et al., 2009, Zhao et al., 2010). In addition, Yang et al. reported that oroxylin A could induce G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells (Yang et al., 2008). Our preliminary experiments demonstrated that oroxylin A could suppress human breast cancer cell MDA-MB-435 invasion through down-regulating the expression of MMP-2 and MMP-9 (Sun et al., 2009), but the molecular mechanisms by which oroxylin A acts on the expression of MMP-2 and MMP-9 and the invasiveness of breast cancer are still undefined and warrant more investigation.

In the present study, we elucidated the potential effects of oroxylin A on the adhesion, invasion, and migration of MDA-MB-231 cells. We also used western blotting, real-time PCR and gelatin zymography to examine the potential of oroxylin A in inhibiting the transcription, expression and activity of MMP-2 and MMP-9. Furthermore, the effect of oroxylin A on the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and PKCδ signaling pathways, which are related to MMP-9 signal transduction, was investigated.