Blocking Palmitoylation of Apelin Receptor Alleviates Morphine Tolerance in Neuropathic Cancer Pain

Neuropathic cancer pain (NCP) is an important symptom in patients with cancer. However, significant analgesic tolerance and other side effects critically hamper the administration of morphine. Protein palmitoylation mediated by the DHHC family may be involved in the glial activation and inflammatory responses underlying organ failure. In this study, we investigated the key role of protein palmitoylation in cancer pain and sought to target palmitoylation to suppress morphine tolerance. We found that long-term use of morphine led to the accumulation of the morphine metabolite, morphine-3-glucuronide, in vivo and activated ERK1/2 and microglia to release inflammatory factors through the apelin receptor APLNR. Palmitoyltransferase ZDHHC9 was upregulated in NCP, and APLNR was palmitylated to protect it from lysosomal degradation and to maintain its stability. We also designed competitive inhibitors of APLNR palmitoylation to inhibit the development of NCP, release of inflammatory factors, and attenuation of morphine tolerance. Therefore, targeting APLNR palmitoylation in combination with morphine is a potent method for cancer pain treatment. Our data provide a basis for the future clinical use of related drugs combined with morphine for the treatment of cancer-related pain.


Cell culture and plasmids
Mouse sarcoma S-180 cells and microglial BV2 cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), where they were characterized by deoxyribonucleic acid (DNA) fingerprinting and isozyme detection.Cell lines were cultured in Dulbecco's modified eagle medium (Gibco BRL, New York, USA) supplemented with 10% fetal bovine serum and 1% (100×) penicillin/streptomycin (Gibco) in the presence of 5% CO2 at 37°C.APLNR (C325, 326A) mutant plasmids were generated by site-directed mutagenesis using the Fast Mutagenesis System (FM111-01) from TransGen Biotech (Beijing, China).All plasmids were verified by DNA sequencing.

For immunoprecipitation, western blotting and immunoprecipitation lysates (20 mM
Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, and leupeptin) (Beyotime, Shanghai, China) with a cocktail (Roche, Basel, Switzerland) were used to prepare protein extracts.To every 500 µg of lysate, 1 µg of the corresponding antibody was added and combined overnight at 4°C.The mixture was incubated with 30 µL protein A/G beads at room temperature for 2 h.After the bonded beads were washed with PBST five times, 30 µL of sodium dodecyl sulfate (SDS) sample buffer was added for western blot analysis.

Immunofluorescence assay
Mice were anesthetized using an intraperitoneal injection of pentobarbital (150 mg/kg) and transcardially perfused with saline solution, followed by 4% paraformaldehyde.
The L4-L6 spinal dorsal horn was then removed, fixed in paraformaldehyde, and dehydrated in 10%, 20%, and 30% sucrose (Sinopharm Chemical Reagent Co. Ltd, Beijing, China.) in succession until they sank and cut into 12-μm sections using a freezing microtome (Leica, Wetzlar, Germany).Sections were blocked with 4% normal goat serum, followed by incubation with primary antibodies: anti-Iba1 antibody (1:500) at 4°C overnight.After being washed with PBST, secondary antibodies labeled AlexaFluor 488 or 555 (Molecular Probes, New York, USA), were incubated at room temperature for 40 min.Slides were observed under a fluorescence microscope (Leica, Wetzlar, Germany).Negative controls were prepared by omitting the primary antibodies.

Cell viability assay
The cells were seeded in 96-well plates at 4,000 cells/well and incubated overnight.A Cell Counting Kit-8 assay (Beyotime, Shanghai, China) was used to detect cell viability.Absorbance was measured at a wavelength of 450 nm.All experiments were performed in triplicate.