Label-Free Dynamic Mass Redistribution Reveals Low-Density, Prosurvival α_1B-Adrenergic Receptors in Human SW480 Colon Carcinoma Cells

Abstract

Small molecules that target the adrenergic family of G protein-coupled receptors (GPCRs) show promising therapeutic efficacy for the treatment of various cancers. In this study, we report that human colon cancer cell line SW480 expresses low-density functional a 1B -adrenergic receptors (ARs) as revealed by label-free dynamic mass redistribution (DMR) signaling technology and confirmed by quantitative reverse-transcriptase polymerase chain reaction analysis. Remarkably, although endogenous a 1B -ARs are not detectable via either [3 H]-prazosin-binding analysis or phosphoinositol hydrolysis assays, their activation leads to robust DMR and enhanced cell viability. We provide pharmacological evidence that stimulation of a 1B -ARs enhances SW480 cell viability without affecting proliferation, whereas stimulating b-ARs diminishes both viability and proliferation of SW480 cells. Our study illustrates the power of label-free DMR technology for identifying and characterizing low-density GPCRs in cells and suggests that drugs targeting both a 1B -and b-ARs may represent valuable smallmolecule therapeutics for the treatment of colon cancer.

Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

“…Next, we examined CHX degradation in SW480 human colon carcinoma cells, which we previously determined to express primarily the α 1B -AR subtype, with minimal to no expression of the α 1A and α 1D subtypes. 17 SNAP-CHX assays reveal that SNAP-α 1B -AR degrades with t 1/2 = 0.77 ± 0.23 h and is ~75% degraded at 24 h ( Fig. 4B,C ).…”

“…We and others have utilized SNAP-tag technology to monitor GPCR protein expression and localization in vitro. 16–25 Here, we investigated the utility of incorporating SNAP-tag technology into traditional CHX-chase assays to permit calculation of GPCR protein half-lives when expressed in cultured human cells. Our experimental workflow involved fusing the SNAP-tag to the N-terminal domain of select GPCRs, transiently transfecting SNAP-GPCR cDNA constructs into HEK293 cells, and then treating cells with 50 µg/mL CHX for 0–24 h. Cells were then lysed and treated with the irreversible SNAP substrate BG-782, subjected to PAGE, and analyzed with LICOR Odyssey NIR quantitative analysis.…”

β2-Adrenoceptor Activation Favor Acquisition of Tumorigenic Properties in Non-Tumorigenic MCF-10A Breast Epithelial Cells

“…In the present study, β 2 -adrenoceptors caused a decrease in cell proliferation, showing the inability of the adrenergic stimulation to promote this cancer hallmark. Previously, it has been shown that adrenoceptors activation can decrease cell proliferation in astrocytoma [104], and in breast [105], melanoma [106], and colon cancer cells [107]. In MCF-10A cells, other authors observed identical results in cell proliferation after β-adrenoceptor activation [31,53].…”

The Neutrophil Dynamic Mass Redistribution Assay as a Medium throughput Primary Cell Screening Assay

“…While this study has concentrated on neutrophil responses, chemokine receptors are known to be expressed in different types of tumor cells, where they can modulate cell invasion, migration and metastasis (Liu et al, 2019;Shang and Li, 2019). As a label-free technique, DMR can be used across a variety of cell types, including cancer cells (Du et al, 2009;Harris et al, 2017).…”