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Characterization of cannabinoid receptors expressed in Ewing sarcoma TC-71 and A-673 cells as potential targets for
Amal M Shoeib1, Azure L Yarbrough2, Benjamin M Ford1
1Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America.
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.
Insights
Cannabinoids induce cancer cell death in Ewing sarcoma by targeting non-canonical cannabinoid receptors (CBRs). These findings highlight atypical CBRs as potential therapeutic targets for Ewing sarcoma treatment.
Area of Science:
- Oncology
- Pharmacology
- Molecular Biology
Background:
- Ewing sarcoma (EWS) is a rare and aggressive bone cancer.
- Cannabinoid receptors (CBRs) are potential targets for anti-cancer drug development.
- Understanding CBR expression and function in EWS is crucial for therapeutic strategies.
Purpose of the Study:
- To characterize cannabinoid receptors (CBRs) in Ewing sarcoma (EWS) cell lines.
- To investigate CBRs as potential therapeutic targets for EWS.
- To explore the anti-cancer effects of cannabinoids on EWS cells.
Main Methods:
- Quantitative reverse transcription PCR (qRT-PCR) to detect CBR mRNA.
- Competitive binding assays to assess CBR affinity.
- G-protein activation and adenylyl cyclase assays to evaluate CBR function.
- Lactate dehydrogenase (LDH) and trypan blue assays to measure cytotoxicity and cell viability.
Main Results:
- CB1 and CB2 receptor (CB1R, CB2R) mRNA were detected in TC-71 EWS cells.
- Expressed CBRs exhibited atypical binding properties, differing from canonical receptors.
- Specific cannabinoids demonstrated high affinity for these non-canonical CBRs.
- Cannabinoids with high affinity reduced EWS cell viability and induced cytotoxicity.
- Similar atypical CBR properties and cannabinoid-induced cytotoxicity were observed in A-673 EWS cells.
Conclusions:
- Cannabinoids induce cytotoxicity in EWS cell lines through non-canonical CBRs.
- These non-canonical CBRs represent a potential therapeutic target for EWS.
- Further research into cannabinoid-based therapies for EWS is warranted.
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