Elsevier

Biochemical Pharmacology

Volume 79, Issue 12, 15 June 2010, Pages 1714-1726
Biochemical Pharmacology

Suppression of PMA-induced tumor cell invasion by dihydroartemisinin via inhibition of PKCα/Raf/MAPKs and NF-κB/AP-1-dependent mechanisms

https://doi.org/10.1016/j.bcp.2010.02.003Get rights and content

Abstract

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently been shown to possess antitumor activity in various cancer cells. However, the effects of DHA in preventing the invasion of cancer cells have not been studied. In the present study, we investigated the inhibitory effects of DHA on tumor invasion and migration and the possible mechanisms involved using human fibrosarcoma HT-1080 cells. DHA reduced PMA-induced activation of MMP-9 and MMP-2 and further inhibited cell invasion and migration. DHA suppressed PMA-enhanced expression of MMP-9 protein, mRNA, and transcriptional activity through suppressing NF-κB and AP-1 activation without changing the level of tissue inhibitor of metalloproteinase (TIMP)-1. DHA also reduced PMA-enhanced MMP-2 expression by suppressing membrane-type 1 MMP (MT1-MMP), but did not alter TIMP-2 levels. DHA-inhibited PMA-induced NF-κB and c-Jun nuclear translocation, which are upstream of PMA-induced MMP-9 expression and invasion. Furthermore, DHA strongly repressed the PMA-induced phosphorylation of Raf/ERK and JNK, which are dependent on the PKCα pathway. In conclusion, we demonstrated that the anti-invasive effects of DHA may occur through inhibition of PKCα/Raf/ERK and JNK phosphorylation and reduction of NF-κB and AP-1 activation, leading to down-regulation of MMP-9 expression. The data presented show that DHA is an effective anti-metastatic agent that functions by down-regulating MMP-9 gene expression.

Graphical abstract

Proposed signal transduction pathways by which PMA induced and DHA-inhibited invasion and migration of HT-1080 cells.

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Introduction

Artemisinin is a sesquiterpene lactone endoperoxide found in the traditional Chinese medicinal plant Artemisia annua[1], [2], and its derivatives are very effective blood schistocidal antimalarials with fewer adverse side effects than other antimalarial drugs. Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is recommended as a first-line antimalarial drug, with low toxicity [3]. In recent years, certain artemisinin derivatives have been shown to inhibit the growth of a limited set of human cancer cell lines [4], [5], such as fibrosarcoma cells [6], breast cancer cells [7], [8], cervical cancer cells [9], leukemia cells, and ovarian cells [10], [11]. Moreover, Jiao et al. also demonstrated that DHA was the most effective and potent agent in inhibiting cell growth of ovarian cancer cells, as compared with artemisinin, artesunate, arteether, artemether, and arteannuin [12]. More importantly, DHA has been shown to be selectively toxic to breast cancer cells, versus normal human breast cells [13], and exerted potent cytotoxic effects on ovarian carcinoma cells, but had minimal effects on non-tumorigenic human ovarian surface epithelial cells [11], suggesting that DHA is well-tolerated and may represent a promising potent therapeutic agent to treat cancers. Consequently, in recent years a hypothesis has been advanced that artemisinin and its derivatives may be useful as anticancer drugs [8]. Although various bioactivity studies of DHA have been carried out, the molecular mechanisms by which DHA acts on the expression of matrix metalloproteinase (MMP)-9 and the invasiveness of HT-1080 are still unclear.

Metastasis and invasion are fundamental properties of malignant cancer cells. An inability to control metastasis and invasion is the leading cause of death in patients with cancer. The control of metastasis and invasion, therefore, represents an important therapeutic target. Tumor invasion and metastasis require increased expression of matrix metalloproteinases (MMPs) [14]. Cell-extracellular matrix (ECM) interactions, disconnection of intercellular adhesion, degradation of the ECM, and the invasion of lymph and blood vessels are important steps in cancer invasion and metastasis [15], [16], [17]. Members of the MMP family are involved in the degradation of ECM, and MMPs have been implicated in malignancy [14], [18]. MMPs are synthesized as preproenzymes and are secreted from cells as proenzymes. Among the human MMPs reported to date, MMP-2 and -9 are the key enzymes involved in degrading Type-I and -IV collagens and ECM [17], [18], [19]. Both MMP-2 and -9, which are abundantly expressed in various malignant tumors [17], contribute to cancer invasion and metastasis [20]. Generally, MMP-2 is constitutive and is over-expressed in highly metastatic tumors, whereas MMP-9 can be stimulated by the inflammatory cytokine tumor necrosis factor (TNF)-α, by the growth factor epidermal growth factor, and by phorbol esters, through activation of different intracellular signaling pathways [21], [22], [23]. Its primary activation occurs on the cell surface and is mediated by membrane-type matrix metalloproteinases (MT-MMPs), such as MT1-MMP [24]. The concerted action of highly expressed MT1-MMP and adequate expression of tissue inhibitor of metalloproteinase (TIMP) leads to activation of MMPs [25], [26].

Mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and protein kinase C (PKC) signaling pathways are the predominant cascades participating in MMP-9 expression [27], [28], [29]. Furthermore, stimulators, such as cytokines and phorbol myristate acetate (PMA), control the expression of MMP-9 by modulating the activation of transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) and through Ras/Raf/MAPKs and PI-3K/AKT signaling pathways [21], [28], [30], because the promoter region of MMP-9 has AP-1 and NF-κB binding sites [31]. NF-κB and AP-1 are well-known transcription factors that regulate the expression of a number of genes, the products of which are involved in metastasis, tumorigenesis, and inflammation [21], [32], [33]. Indeed, NF-κB and AP-1 are key transcription factors in the activation of genes encoding inflammatory cytokines, such as TNF-α and IL-1β[33], [34], [35]. NF-κB can also induce the activation of MMP-9 and COX-2 [32], [36]. Several studies have indicated that inhibition of MMP expression or enzyme activity can be used as early targets for preventing cancer metastasis [17], [20]. Thus, agents possessing the ability to suppress the expression of MMP-2 or -9 are worthy of investigation with regard to cancer cell invasion and metastasis.

In this research, we studied the effects of DHA on PMA-induced MMP expression and explored the underlying upstream signaling molecular mechanisms. We found that DHA significantly suppressed MMP-9 gene expression by blocking the conventional PKCα/Raf/MAPKs and NF-κB/AP-1 signaling pathways, consequently reducing invasion and metastasis of HT-1080 cells.

Section snippets

Materials

Dihydroartemisinin (DHA), curcumin, and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma Chemical (St. Louis, MO). NF-κB activation inhibitor (JSH-23), Gö6976, U0126, SB203580, and SP600125 were purchased from Calbiochem (La Jolla, CA). The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]-based colorimetric assay kit was purchased from Roche (Indianapolis, IN). RPMI1640, fetal bovine serum (FBS), sodium pyruvate, and Trizol were from Gibco BRL (Grand Island, NY).

DHA suppresses PMA-induced invasion and migration of HT-1080 cells through inhibition of MMP-9 expression

Prior to investigating the pharmacological potential of DHA on PMA-induced MMP activity, we first determined the dose dependence of the cytotoxic effects of DHA in the absence or presence of PMA (30 nM) for 24 h in HT-1080 cells using the MTT assay. DHA at concentrations lower than 50 μM had no cytotoxic effect on the cells and DHA at 100 μM showed about a 20–25% decreases in cell viability in the absence or presence of PMA (Fig. 1B). We next used a gelatin zymography assay to investigate the

Discussion

Artemisinin is a sesquiterpene lactone peroxide containing an endoperoxide moiety. DHA is the main active metabolite of artemisinin derivatives and is more water-soluble and a more effective antimalarial than artemisinin. After the discovery of artemisinin and its derivatives, especially DHA, as a novel and promising treatment for cancer, many studies have investigated the use of DHA in the treatment of cancers [12], [13], [47], [48]. It is well established that tumor cell migration and

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grants from the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by Ministry of Education, Science and Technology (2009-0093815) and the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs (A084869), Republic of Korea.

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