Abstract
Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to a broad range of DNA-damaging chemotherapeutics. We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16:4(n-3). Studies that used splenocytes from GPR120-deficient mice have confirmed this conclusion. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance-inducing lipid mediator, lysophosphatidylcholine(24:1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.—Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.
Highlights
ABBREVIATIONS: 12-S-HHT, 12-S-hydroxy-5,8,10-heptadecatrienoic acid; 16:4(n-3), hexadeca-4,7,10,13-tetraenoic acid; AACOCF3, arachidonyl trifluoromethyl ketone; BSA, bovine serum albumin; eYFP, enhanced yellow fluorescent protein; FACS, fluorescence-activated cell sorting; H2AX, histone 2A family member X; HRP, horseradish peroxidase; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; aLA, a-linolenic acid; NLUC, nanoluciferase; PC, phosphatidylcholine; sCM, splenic conditioned medium; platinuminduced fatty acids (PIFAs), platinum-induced fatty acid; Tris-buffered saline/ Tween 20 (TBST), Tris-buffered saline/Tween 20
We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs)
By using combinations of selective pharmacologic activation and inhibition of G protein-coupled receptor 40 (GPR40) and GPR120 in concert with splenocytes isolated from both wild-type and GPR120-deficient mice, we show that effects of 16:4(n-3) are induced via GPR120 and that activation of this receptor results in a signaling cascade within splenocytes that involves cytosolic PLA2-mediated generation and release of a specific isoform of lysophosphatidylcholine (LPC), which acts as the ultimate inducer of chemoresistance
Summary
16:4(n-3) was isolated from Ulva pertusa as previously described [23]. GW1100 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Splenocytes were incubated with F4/80-FITC and CD11b-APC (concentration 1 ml/1 3 107 cells/100 ml) or corresponding isotype controls (FITC and APC; both from eBioscience) in FACS buffer for 30 min on ice. F4/80+/CD11blow, F4/80+/ CD11bhigh, F4/802/CD11bhigh and F4/802/CD11b2 populations were sorted by using a FACSAria III (BD Bioscience, Brea, CA, USA). F4/80+ splenocytes were lysed after incubating them for the indicated times with 25 nM 16:4(n-3) [or 25 nM 16:4(n-3) and a 10-min preincubation of 10 mM AH-7614], and cPLA2 or PLA1 activity was measured by using EnzChek PLA2 or EnzChek PLA1 assay kit, respectively (Thermo Fisher Scientific), according to manufacturer protocol. Test compounds were added at the specified concentration, and cells incubated for a final 5 min before measuring luminescent emission at 530 and 465 nm using a ClarioStar plate reader (BMG Labtech, Cary, NC, USA).
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