Sinomenine inhibits breast cancer cell invasion and migration by suppressing NF-κB activation mediated by IL-4/miR-324-5p/CUEDC2 axis

Highlights

• Sinomenine reduced invasion and migration of MDA-MB-231 and 4T1 breast cancer cells.

• Sinomenine enhanced combination of NF-κB with IκB and blocked NF-κB activation.

• Sinomenine promoted CUEDC2 expression and suppressed IκB kinase phosphorylation.

• Sinomenine reduced IL-4 and miR-324-5p levels.

• MiR-324-5p inhibited the suppression of invasion and metastasis by sinomenine.

Abstract

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a vital transcription factor that regulates multiple important biological processes, including the epithelial–mesenchymal transition (EMT) and metastasis of breast cancer. Sinomenine is an isoquinoline well known for its remarkable curative effect on rheumatic and arthritic diseases and can induce apoptosis of several cancer cell types. Recently, sinomenine was reported as a tumor suppressor via inhibiting cell proliferation and inducing apoptosis. However, the role and mechanism of sinomenine in invasion and metastasis of breast cancer are largely unknown. Here, we report that sinomenine suppressed the invasion and migration of MDA-MB-231 and 4T1 breast cancer cells in a dose-dependent manner. We detected binding of NF-κB to the inhibitor of NF-κB (IκB) after the MDA-MB-231 cells were treated with 0.25, 0.5, and 1 mM sinomenine. Co-IP analysis revealed that sinomenine enhanced the binding of NF-κB and IκB in a dose-dependent manner, suggesting that sinomenine had an effect on inactivation of NF-κB. Western blotting and ELISA approaches indicated that the suppression effect was closely associated with the phosphorylation of IκB kinase (IKK) and its negative regulator CUEDC2. Sinomenine treatment decreased miR-324-5p expression, thus increased the level of its target gene CUEDC2, and then blocked the phosphorylation of IKK through altering the upstream axis. Finally, transfection of a miR-324-5p mimic inhibited the suppression of invasion and metastasis of MDA-MB-231 and 4T1 cell by sinomenine, providing evidence that sinomenine treatment suppressed breast cancer cell invasion and metastasis via regulation of the IL4/miR-324-5p/CUEDC2 axis. Our findings reveal a novel mechanism by which sinomenine suppresses cancer cell invasion and metastasis, i.e., blocking NF-κB activation.

Introduction

Breast cancer is a type of malignant tumor that occurs in the mammary gland epithelial tissue and is the greatest cancerous threat to women's health [1]. The incidence rate of breast cancer has exhibited a global upward trend since 1980s [2], [3], [4], [5], [6]. Breast cancer is occasionally invasive or metastatic, and causes a high fatality rate once it spreads. In 2013, the Chinese Tumor Registry Annual Reports showed that the incidence and mortality of breast cancer ranked second among all the malignant tumors [7], [8]. Exploring the mechanism of metastasis and developing drugs to inhibit metastasis are important to reduce the mortality caused by breast cancer.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a vital transcription factor with a variety of target genes that are involved in the regulation of multiple important biological processes, including cell proliferation and apoptosis, enzyme synthesis, and cell cycle control, as well as cell adhesion and secretion [9], [10]. Angiogenesis, reduction of cell adhesion, and a change in the extracellular matrix components are the preferred microenvironment for tumor metastasis [11], [12], [13], [14]. In the last decade, studies revealed that NF-κB is essential for invasion and metastasis of some cancers [15], [16]. In breast cancer, NF-κB is required for the epithelial–mesenchymal transition (EMT), which is considered to be closely identified with invasion and metastasis [17], [18].

NF-κB is present in cells in an inactive state in which it is sequestered in dimers by IκB (inhibitors of κB) in the cytoplasm until cellular stimuli trigger its inducers [19]. The induction of NF-κB activation consists of a series of signal transductions, which are usually summarized as being canonical and non-canonical pathways [20]. The final effects of these pathways are IκB degradation and NF-κB release, which then translocates into the nuclear. During these processes, activation of a kinase called IκB kinase (IKK) is required [21]. Therefore, a key mechanism of keeping NF-κB inactive is to block the activation of IKK. CUE domain-containing 2 (CUEDC2) was recently proven to be an IKK activation inhibitor through recruitment of the phosphatase PP1 and by blocking phosphorylation of IKK [22]. This function of dephosphorylating IKK highlighted CUEDC2 as an important suppressor of NF-κB-mediated tumorigenesis and progression. Moreover, upstream regulators of CUEDC2, such as miR-324-5p [23], have also attracted attentions.

Sinomenine is an isoquinoline named after its extraction from the Chinese medicinal plant Sinomenium acutum. Sinomenine is well known for its remarkable curative effect on rheumatic and arthritic diseases [24]. In recent years, many reports have suggested that sinomenine has an anti-cancer function in breast and many other cancers due to its role in cell proliferation suppression and apoptosis promotion [25], [26]. Furthermore, sinomenine was found to reduce invasion and migration ability of several types of cells [27], [28]. However, the mechanism is still not fully clarified.

In this study, MDA-MB-231 and 4T1 breast cancer cell lines were incubated with sinomenine, and their invasion and migration capacity were examined. Sinomenine significantly suppressed the invasion and migration capacity of MDA-MB-231 and 4T1 cell lines. Sinomenine inhibited phosphorylation of IKK and activation of NF-κB through mediating the upstream IL4/miR-324-5p/CUEDC2 axis.