Anti-cancer and analgesic effects of resolvin D2 in oral squamous cell carcinoma
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.
Section snippets
Cell culture of human oral SCC, normal oral keratinocytes, and dysplastic oral keratinocytes
HSC-3 cells (Japanese Collection of Research Bioresources, passage 4–10) were grown in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing antibiotic (penicillin/streptomycin, 10 U/mL) and 10% fetal bovine serum (FBS). Premalignant dysplastic oral keratinocytes (DOKs, Sigma Aldrich, passage 4–8) were cultured in DMEM with 10% FBS and 5 μg/ml hydrocortisone. Normal oral keratinocytes (NOKs, Lifeline Cell Tech, passage 4–8) were grown in Lifeline® DermaLife K Medium. All cells were
RvD1 and RvD2 receptor mRNAs were downregulated in HSC-3 cells
RvD1 actions are mediated by two G protein-coupled receptors (GPCRs), ALX/FPR2 and GPR32. RvD2 acts on a GPCR called GPR18. We found that both GPR32 and GPR18 were significantly downregulated in HSC-3 cells compared to NOKs (Fig. 1A). GPR32 was significantly downregulated in pre-cancer DOKs compared to NOKs (Fig. 1B). ALX/FPR2 expression was low in NOKs (∼37 CT), barely detectable in DOKs (∼39 CT) and undetectable in HSC-3 (>40 CT, data not shown).
RvD1 inhibited cancer growth in vitro but not in vivo
In the real-time cell analyzer, we observed a
Discussion
In the present study, we tested the hypothesis that endogenous lipid mediators (RvD1 and RvD2) involved in the resolution phase of inflammation reduced oral cancer progression and pain. We found that oral SCC cells had lower mRNA expression for receptors of both RvD1 and RvD2 relative to NOKs; we inferred from these results that the endogenous pro-resolution pathway in oral SCC was suppressed. We found that RvD2 exhibited an anti-proliferative and anti-inflammatory profile in vitro. In vivo,
Conflicts of interest
No potential conflicts of interest were disclosed.
Contributors
All authors contributed to and have approved the final manuscript. Yi Ye designed and performed the experiments, analyzed data, and supervised the project. Nicole N. Scheff, Daniel Bernabé, Elizabeth Salvo, Ratna Veeramachaneni, Kentaro Ono, Dan T. Viet, and Chi T. Viet performed experiments and analyzed data. Cheng Liu performed histopathological examination of mouse tumor tissues. Yi Ye, Nicole N. Scheff, John C. Dolan, Brian L. Schmidt wrote, reviewed, and revised the manuscript.
Funding
This work was supported by grants from the International Association for the Study of Pain (IASP) Young Investigator Award (Y. Ye), New York University Whitehead Fellowship for Young Investigators (Y. Ye), and NIH/DCR R01 DE019796 (B.L. Schmidt).
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