Characterization of cannabinoid receptors expressed in Ewing sarcoma TC-71 and A-673 cells as potential targets for anti-cancer drug development

Life Sci. 2021 Nov 15:285:119993. doi: 10.1016/j.lfs.2021.119993. Epub 2021 Sep 28.

Abstract

Aims: Characterizing cannabinoid receptors (CBRs) expressed in Ewing sarcoma (EWS) cell lines as potential targets for anti-cancer drug development.

Main methods: CBR affinity and function were examined by competitive binding and G-protein activation, respectively. Cannabinoid-mediated cytotoxicity and cell viability were evaluated by LDH, and trypan blue assays, respectively.

Key findings: qRT-PCR detected CB1 (CB1R) and CB2 receptor (CB2R) mRNA in TC-71 cells. However, binding screens revealed that CBRs expressed exhibit atypical properties relative to canonical receptors, because specific binding in TC-71 could only be demonstrated by the established non-selective CB1/CB2R radioligand [3H]WIN-55,212-2, but not CB1/CB2R radioligand [3H]CP-55,940. Homologous receptor binding demonstrated that [3H]WIN-55,212-2 binds to a single site with nanomolar affinity, expressed at high density. Further support for non-canonical CBRs expression is provided by subsequent binding screens, revealing that only 9 out of 28 well-characterized cannabinoids with high affinity for canonical CB1 and/or CB2Rs were able to displace [3H]WIN-55,212-2, whereas two ligands enhanced [3H]WIN-55,212-2 binding. Five cannabinoids producing the greatest [3H]WIN-55,212-2 displacement exhibited high nanomolar affinity (Ki) for expressed receptors. G-protein modulation and adenylyl cyclase assays further indicate that these CBRs exhibit distinct signaling/functional profiles compared to canonical CBRs. Importantly, cannabinoids with the highest affinity for non-canonical CBRs reduced TC-71 viability and induced cytotoxicity in a time-dependent manner. Studies in a second EWS cell line (A-673) showed similar atypical binding properties of expressed CBRs, and cannabinoid treatment produced cytotoxicity.

Significance: Cannabinoids induce cytotoxicity in EWS cell lines via non-canonical CBRs, which might be a potential therapeutic target to treat EWS.

Keywords: Cannabidiol; Cannabinoid receptors; Cell viability; Cytotoxicity; Ewing sarcoma; WIN-55,212-2.

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Benzoxazines / pharmacology*
  • Binding, Competitive
  • Cannabinoids / pharmacology*
  • Cell Line, Tumor
  • Cytotoxins / pharmacology
  • Drug Development
  • Humans
  • Ligands
  • Morpholines / pharmacology*
  • Naphthalenes / pharmacology*
  • Receptor, Cannabinoid, CB1 / agonists
  • Receptor, Cannabinoid, CB1 / metabolism*
  • Receptor, Cannabinoid, CB2 / agonists
  • Receptor, Cannabinoid, CB2 / metabolism*
  • Sarcoma, Ewing / metabolism*

Substances

  • Antineoplastic Agents
  • Benzoxazines
  • Cannabinoids
  • Cytotoxins
  • Ligands
  • Morpholines
  • Naphthalenes
  • Receptor, Cannabinoid, CB1
  • Receptor, Cannabinoid, CB2
  • (3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone