Effects of regulator of G protein signaling (RGS) proteins on μ and δ opioid receptors were investigated in HEK293 cells. Co-expression of RGS1, RGS2, RGS4, RGS9, RGS10 or RGS19 (Gα-interacting protein (GAIP)) significantly reduced [Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol]-Enkephalin (DAMGO)-induced inhibition of adenylyl cyclase (AC) mediated by μ opioid receptor, but only RGS9 decreased the effects of [Tyr-D-Pen-Gly-p-Chloro-Phe-D-Pen]-Enkephalin (DPDPE) mediated by δ opioid receptor. When C-tails of the receptors were exchanged (μ/δC and δ/μC chimeras), RGS proteins decreased δ/μC-mediated AC inhibition, but none had significant effects on that via μ/δC receptor. Thus, the C-terminal domains of the receptors are critical for the differential effects of RGS proteins, which may be due to differences in receptor -G protein -RGS protein interactions in signaling complexes.
BackgroundCancer immunotherapy has generated significant excitement, mainly as a result of the development of immune checkpoint inhibitors. The blockade of PD-1 or its ligand with antibodies has resulted in impressive clinical efficacy. However, a subset of patients does not respond to biologic therapeutics, and another subset suffers from severe immune-related adverse events in certain cases. The modulation of the immune system with small molecules might yield surprising benefits.MethodsCD8+ cells were obtained through a magnetic cell sorting system (MACS), and their capabilities for IFN-γ release and PD-1 expression were analyzed. The in vitro effects of drugs were studied in a coculture system of tumor cells and activated CD8+ cells. We further isolated the primary tumor cells in tumor-bearing mice treated with CAI, DMF, 1-MT or a combination (CAI and DMF/CAI and 1-MT) and analyzed the percentages of CD8+ T cells and PD-1+CD8+ T cells among TILs. The selective anti-tumor immune reactions of the two drug combinations were confirmed in a coculture system consisting of B16-OVA cells and OVA-specific CTLs derived from OT-1 transgenic mice. The anti-tumor effects of the single drugs or combined therapies were assessed according to their capability to slow tumor growth and extend the life span of tumor-bearing mice, and they were compared with the effects of PD-1 antibody.ResultsCAI increased IFN-γ release from activated T cells, which might strengthen the anti-proliferative and anti-metastatic effects on cancer cells. However, CAI also stimulated IDO1-Kyn metabolic circuitry in the tumor microenvironment and facilitated tumor cell immune evasion. Combining CAI with 1-MT or DMF disrupted PD-1 expression and promoted IFN-γ production in CD8+ T cells, and it also increased T lymphocyte infiltration in the tumor microenvironment, inhibited tumor growth and prolonged the life spans of tumor-bearing mice.ConclusionInhibitors of the IDO1-Kyn-AhR pathway could abolish the negative effects of CAI on CD8+ T cells and result in complementary and beneficial anti-tumor immune effects. The combination of CAI with 1-MT or DMF greatly augmented the ability of CD8+ T cells to kill malignant cells and showed a strong anti-cancer capability that was superior to that of either of the single agents was is comparable with that of anti-PD-1 antibody. The combinations of small molecules utilized in this study may serve as valuable new immunotherapy strategies for cancer treatment.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0725-7) contains supplementary material, which is available to authorized users.
BACKGROUND AND PURPOSEPro-inflammatory cytokines are important in rheumatoid arthritis (RA) and their production is mainly regulated by NF-κB and inflammasomes. Carboxyamidotriazole (CAI) exhibits potent anti-inflammatory activities by decreasing cytokines. Here, we have investigated NACHT, LRR and PYD domains-containing protein (NALP) inflammasomes in a rat model of RA and explored the therapeutic effects of CAI in this model and the involvement of NF-κB and inflammasomes in the actions of CAI.
EXPERIMENTAL APPROACHThe anti-arthritic effects of CAI were assessed in the adjuvant arthritis (AA) model in rats, using radiological and histological techniques. NALP1 and NALP3 inflammasomes, NF-κB pathway and pro-inflammatory cytokines levels were measured with Western blots, immunohistochemistry and ELISA.
KEY RESULTSCAI decreased the arthritis index, improved radiological and histological changes, and reduced synovial IL-1β, IL-6, IL-18 and TNF-α levels in rats with AA. Compared with normal rats, the 70 kDa NALP1 isoform was up-regulated, NALP3 was down-regulated, and levels of the 165 kDa NALP1 isoform and the adaptor protein ASC were unchanged in synovial tissue from AA rats. CAI reduced the 70 kDa NALP1 isoform and restored NALP3 levels in AA rats; CAI inhibited caspase-1 activation in AA synovial tissue, but not its enzymic activity in vitro. In addition, CAI reduced expression of p65 NF-κB subunit and IκBα phosphorylation and degradation in AA rats.
CONCLUSION AND IMPLICATIONSNALP1 inflammasomes were activated in synovial tissues from AA rats and appeared to be a novel therapeutic target for RA. CAI could have therapeutic value in RA by inhibiting activation of NF-κB and NALP1 inflammasomes and by decreasing pro-inflammatory cytokines.
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