Anti-cancer and analgesic effects of resolvin D2 in oral squamous cell carcinoma

Highlights

• RvD2 inhibits oral squamous cell carcinoma growth.

• RvD2 counter-regulates cancer-derived cytokine/chemokines IL-6 and CXCL10.

• RvD2 transiently inhibits mechanical and thermal pain and immune cell infiltration.

• RvD2 might enhance endogenous clearance of tumor necrosis.

• RvD1 exhibits long-lasting attenuation of thermal pain.

Abstract

Oral cancer is often painful and lethal. Oral cancer progression and pain may result from shared pathways that involve unresolved inflammation and elevated levels of pro-inflammatory cytokines. Resolvin D-series (RvDs) are endogenous lipid mediators derived from omega-3 fatty acids that exhibit pro-resolution and anti-inflammatory actions. These mediators have recently emerged as a novel class of therapeutics for diseases that involve inflammation; the specific roles of RvDs in oral cancer and associated pain are not defined. The present study investigated the potential of RvDs (RvD1 and RvD2) to treat oral cancer and alleviate oral cancer pain. We found down-regulated mRNA levels of GPR18 and GPR32 (which code for receptors RvD1 and RvD2) in oral cancer cells. Both RvD1 and RvD2 inhibited oral cancer proliferation in vitro. Using two validated mouse oral squamous cell carcinoma xenograft models, we found that RvD2, the more potent anti-inflammatory lipid mediator, significantly reduced tumor size. The mechanism of this action might involve suppression of IL-6, C-X-C motif chemokine 10 (CXCL10), and reduction of tumor necrosis. RvD2 generated short-lasting analgesia in xenograft cancer models, which coincided with decreased neutrophil infiltration and myeloperoxidase activity. Using a cancer supernatant model, we demonstrated that RvD2 reduced cancer-derived cytokines/chemokines (TNF-α, IL-6, CXCL10, and MCP-1), cancer mediator-induced CD11b⁺Ly6G⁻ myeloid cells, and nociception. We infer from our results that manipulation of the endogenous pro-resolution pathway might provide a novel approach to improve oral cancer and cancer pain treatment.

Introduction

Oral squamous cell carcinoma (SCC) is the most common oral malignancy (Wu et al., 2011). Survival of oral SCC patients has modestly improved in the past few decades; recurrence is common (Hunter et al., 2005; Ran and Yang, 2017). Approximately half of all oral SCC patients treated with surgery, chemotherapy, or radiation therapy will be cured. Outside of survival, pain is the primary concern of oral cancer patients (Benoliel et al., 2007; Epstein et al., 2007; Viet and Schmidt, 2012). Oral cancer pain usually becomes more severe with disease progression (Benoliel et al., 2007). Terminal oral SCC patients often experience excruciating pain during the final months of life (Benoliel et al., 2007). Ineffective pain control correlates positively with poor outcome and diminished quality of life in oral SCC patients (Reyes-Gibby et al., 2014; Viet and Schmidt, 2012).

Chronic inflammation is a hallmark of oral SCC (Choi and Myers, 2008; Feller et al., 2013a; Patel et al., 2016; Wu et al., 2011). Acute inflammation is typically self-limited, initiated by pro-inflammatory cytokines and actively resolved by specialized lipid mediators (i.e., pro-resolution, anti-inflammatory mediators) that return inflamed tissues to homeostasis (Ji et al., 2011; Prevete et al., 2017a; Serhan et al., 2008). These specialized pro-resolving mediators are derived from ω-3 or ω-6 essential polyunsaturated fatty acids (PUFA), through the activity of lipoxygenases (Serhan et al., 2002). A potentiated pro-inflammatory response or a suppressed anti-inflammatory response can impair resolution and lead to chronic inflammation; inflammation increases cancer proliferation, progression, and metastasis (Feller et al., 2013a; Prevete et al., 2017a). The well-known pro-inflammatory pathway associated with oral SCC involves cyclooxygenase-2 (COX-2) (Feller et al., 2013b; Hunter et al., 2005; Pandey et al., 2008), the inducible enzyme that converts arachidonic acid to prostaglandins. COX-2 inhibitors, however, generate slight or no improvement in oral premalignant lesions (Feller et al., 2013a; Hunter et al., 2005; Papadimitrakopoulou et al., 2008; Wirth et al., 2008). COX-2 inhibitors slightly reduce oral cancer pain in animal models (Harano et al., 2010). Moreover, COX-2 inhibitors might prolong inflammation by blocking synthesis of endogenous pro-resolution mediators (Ji et al., 2011; Serhan et al., 2002, 2008). The role of counter-regulatory mechanisms (i.e., the pro-resolution pathway) has never been studied in the context of oral SCC progression and pain.

Resolvins, such as resolvin D1 (RvD1) and resolvin D2 (RvD2), represent a family of pro-resolution mediators naturally synthesized from docosahexaenoic acid (DHA) (Serhan et al., 2002, 2008). They exert potent pro-resolving and anti-inflammatory actions by regulating immune cell migration and infiltration, removing cell debris and apoptotic cells, and through inhibition of pro-inflammatory cytokines synthesis (Ji et al., 2011; Serhan, 2014; Sulciner et al., 2017). The benefits of RvDs have been reported in many disease models associated with inflammation including cancer of the lung, liver, pancreas, stomach, and colon, as well as in models of inflammatory pain and postoperative pain (Halder et al., 2015; Huang et al., 2011; Ji et al., 2011; Kuang et al., 2016; Lee et al., 2013a; Prevete et al., 2017b; Serhan, 2014; Wang and Strichartz, 2017; Xu et al., 2010). RvD1 and RvD2 attenuate or alleviate pain through inhibition of transient receptor potential (TRP) ion channels and reduction of inflammatory mediators including tumor necrosis factor alpha (TNF-α) and IL-6 (Claria et al., 2012; Ji et al., 2011; Xu et al., 2010). In addition, these resolvins inhibit glial activation and spinal cord synaptic transmission (Ji et al., 2011). Low doses of RvD1 and RvD2 are sufficient for treatment in animal models of inflammation and are not immunosuppressive; no side effects have been observed (Ji et al., 2011; Prevete et al., 2017a; Sulciner et al., 2017). Accordingly, RvD1 and RvD2 are attractive therapeutic candidates for cancer and cancer symptom treatment.

In the present study we hypothesized that RvD1 and RvD2 counter-regulate the pro-inflammatory microenvironment driven by oral SCC. Using cell culture and mouse models of oral cancer we examined the effect of RvD1 and RvD2 on cytokine production, immune cell infiltration, tumor growth, and nociceptive behaviors. We provided evidence that RvD2 significantly inhibited tumor growth. We proposed a mechanism of action that includes attenuated tumor-promoting cytokines and hypoxia-induced necrosis. RvD2 alleviated acute pain caused by cancer mediators and transiently reduced chronic cancer pain.