Thymoquinone suppresses metastasis of melanoma cells by inhibition of NLRP3 inflammasome

https://doi.org/10.1016/j.taap.2013.03.027Get rights and content

Highlights

  • Thymoquinone causes inhibition of migration of melanoma cells.

  • Thymoquinone causes inhibition of metastasis in vivo.

  • Thymoquinone causes inhibition of migration by activation of NLRP3 inflammasome.

Abstract

The inflammasome is a multi-protein complex which when activated regulates caspase-1 activation and IL-1β and IL-18 secretion. The NLRP3 (NACHT, LRR, and pyrin domain-containing protein 3) inflammasome is constitutively assembled and activated in human melanoma cells. We have examined the inhibitory effect of thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone), a major ingredient of black seed obtained from the plant Nigella sativa on metastatic human (A375) and mouse (B16F10) melanoma cell lines. We have assessed whether thymoquinone inhibits metastasis of melanoma cells by targeting NLRP3 subunit of inflammasomes. Using an in vitro cell migration assay, we found that thymoquinone inhibited the migration of both human and mouse melanoma cells. The inhibitory effect of thymoquinone on metastasis was also observed in vivo in B16F10 mouse melanoma model. The inhibition of migration of melanoma cells by thymoquinone was accompanied by a decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1. Inactivation of caspase-1 by thymoquinone resulted in inhibition of IL-1β and IL-18. Treatment of mouse melanoma cells with thymoquinone also inhibited NF-κB activity. Furthermore, inhibition of reactive oxygen species (ROS) by thymoquinone resulted in partial inactivation of NLRP3 inflammasome. Thus, thymoquinone exerts its inhibitory effect on migration of human and mouse melanoma cells by inhibition of NLRP3 inflammasome. Thus, our results indicate that thymoquinone can be a potential immunotherapeutic agent not only as an adjuvant therapy for melanoma, but also, in the control and prevention of metastatic melanoma.

Introduction

Melanoma is the most dangerous form of skin tumor and causes 90% of skin cancer mortality (Garbe et al., 2012). There are some treatment options for primary melanoma, but very few options are available for the prevention and control of metastasis. Various chemokines and cytokines have been identified that play a role in promoting tumor invasion in several cancers including melanoma (Opdenakker and Van Damme, 1992, Raman et al., 2007). Therefore, investigation on immune regulation of melanoma may unveil a novel therapeutic agent in prevention and control of metastatic melanoma. Metastasis is a multi-step complex process, which includes a series of events like cellular adhesion to the basement membrane, invasion through the basement membrane, transfer via the circulation, extravasation, and proliferation at a distant site (Fidler and Hart, 1982). Thus, prevention of tumor metastasis is a crucial step to comfort cancer patients and improve their quality of life.

The nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family detects microbial components in the cytosol and triggers the assembly of large caspase-1-activating complexes termed inflammasomes (Lamkanfi and Dixit, 2009, Lamkanfi, 2011). NLRs interacts with ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD), which cleaves and activates caspase-1. Caspase-1 mediates the conversion of pro- interleukin (IL)-1β to secreted and active form of IL-1β in human melanoma cells (Okamoto et al., 2010). Secreting IL-1β into the tumor milieu induces several angiogenic factors from tumor and stromal cells and thus promotes tumor growth through hyperneovascularization (Saijo et al., 2002). Among the two best characterized inflammasomes are NLRP (NACHT, LRR, and pyrin domain-containing protein) 1 and NLRP3. NLRP3 is constitutively activated and leads to auto-inflammation characterized by sustained local and systemic inflammation mediated by IL-1β (Okamoto et al., 2010). NLRP1 does not require ASC for caspase-1 activation as it contains CARD, which plays a role in directly activating caspase-1, leading to cleavage of pro IL-1β to form active IL-1β (Faustin et al., 2007). Recent studies have revealed that, in primary and metastatic melanoma, knockdown of ASC inhibited inflammasome-mediated caspase-1 activity and IL-1β secretion (Liu et al., 2012).

Thymoquinone is the active constituent found in the crude extracts of the seeds of Nigella sativa, and has been shown to have anti-oxidant/anti-inflammatory efficacy in models of asthma, diabetes, encephalomyelitis, neurodegeneration, and carcinogenesis (Abdelmeguid et al., 2010, Kanter, 2008, Woo et al., 2012). Studies have shown that thymoquinone exerts its anti-neoplastic effect(s) through different modes of action, that is, by inhibition of cell proliferation, induction of apoptosis, cell cycle arrest, generation of reactive oxygen species (ROS), and inhibition of metastasis and angiogenesis (Banerjee et al., 2010). Although the anti-metastatic effects of thymoquinone have been reported in numerous studies, the exact mechanism of action is not well understood.

In this study, we have investigated the doses of thymoquinone that inhibit migration of melanoma cells using in vitro and in vivo models. We discovered that thymoquinone inhibited caspase-1 activity, decreased IL-1β and IL-18 secretion, and inhibited migration of melanoma cells by targeting NLRP3 subunit of inflammasomes, thereby causing a decrease in IL-1β secretion as well as inhibition of NF-κB, and hence suppressing growth and migration of melanoma cells.

Section snippets

Animals and reagents

Wild-type female C57BL/6 (WT) mice 6–8 weeks of age were purchased from National Cancer Institute (Frederick, MD). All animal procedures were performed according to the National Institutes of Health guidelines under protocols approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Thymoquinone was purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mouse NLRP3 antibody was purchased from R&D systems (Minneapolis, MN), NF-kB activity kit was

Thymoquinone inhibits proliferation of human and mouse metastatic melanoma cells in vitro

Human melanoma (A375) and mouse melanoma (B16F10) cells were subjected to treatment with varying concentration of thymoquinone (2, 5, 10, 20, 40, 80, 100, 120 and 150 μM) at various time points, which showed decreased proliferation of cells in a dose-dependent manner. Lower concentration of thymoquinone (up to 40 μM) does not inhibit proliferation significantly even up to 72 h, but higher concentration of thymoquinone (beyond 40 μM) revealed a significant inhibition of proliferation potential of

Discussion

Metastasis in the advanced stage of melanoma increases the severity of the disease, decreases the survival of patients and also hampers the prognosis of cancer therapy (Mackie et al., 2009). NLRP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 which leads to the auto-inflammation by processing and secretion of the pro-inflammatory cytokines IL-1β and IL-18 in melanoma cells (van de Veerdonk et al., 2011). The role of activated NLRP3 inflammasome in innate and

Conflict of interest disclosure

None of the authors have a conflict of interest.

Acknowledgment

This work was partially supported by the Department of Defense New Investigator Award W81XWH-10-1-0763 to NY.

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