β‐Adrenergic receptor‐biased β‐blockers for skin cancer prevention

Abstract

Skin cancer, consisting of non‐melanoma skin cancer (NMSC, major subtypes basal and squamous cell carcinoma) and melanoma, is the most common cancer type in the US. These cancers, especially NMSC, have an enormous impact on health care costs. Although ultraviolet radiation from sunlight has been a known cause for NMSC, use of sunscreen as primary prevention has proven inadequate in reducing its incidence. Another risk factor for skin cancer is chronic stress that contributes to carcinogenesis possibly by suppressing immune function and inducing DNA damage. Catecholamines, including norepinephrine and epinephrine, are the primary molecules triggering the body's “fight or flight” response to stress, through the activation of the β‐adrenergic receptors (β‐ARs). Recent clinical and epidemiological studies provide evidence linking the use of β‐AR antagonists (i.e., β‐blockers) with reduced risk of cancer. Our previous study showed that the receptor subtype nonselective β‐blocker carvedilol prevented skin cancer in vitro and in vivo. The objective of the present study is to determine whether the effect observed with carvedilol is a drug specific effect or a general β‐blocker effect. The JB6 P+ cell line is an established chemoprevention model that is non‐cancerous yet transformable by a number of factors including epidermal growth factor (EGF). Thus, 16 commonly prescribed β‐blockers as well as isoproterenol, a full agonist at the β2‐AR, were examined for their ability to prevent EGF‐mediated colony formation of JB6 P+ cells in soft agar. For each compound, sulforhodamine B (SRB) assays were conducted to ensure that the observed results were not due to cytotoxicity. Interestingly, it was observed that GRK/β‐arrestin biased agonist β‐blockers (carvedilol, nebivolol, and alprenolol) showed strong inhibitory activity on colony formation (IC50s < 1 mM). However, most non‐biased β‐blockers (partial, neutral and inverse agonists) failed to inhibit colony formation at concentrations lower than 1 mM. Since it is known that biased agonists trigger β‐arrestin‐dependent ERK1/2 activation, we confirmed that carvedilol, similar to EGF and isoproterenol, was able to induce time‐dependent ERK1/2 activation in JB6 P+ cells. The differential signaling profiles for JB6 P+ cells treated with EGF or carvedilol for 15 min were identified using the Phospho Explorer Antibody Microarray containing 1318 site‐specific and phospho‐specific antibodies from over 30 signaling pathways. Array data analysis suggested that the levels of phosphorylated protein components of NF‐κB and AP‐1 pathways were induced by EGF, which was attenuated by carvedilol. Antibody array results were validated by examining the NF‐κB and AP‐1 promoter activities using dual‐luciferase reporter assays in HEK293 cells. Thus, not all β‐blockers possess chemopreventive properties, only those that were reported as β‐AR biased agonists demonstrated a high chemopreventive efficacy. These results suggest that the unique GRK/β‐arrestin signaling induced by the biased β‐blockers such as carvedilol may contribute to their special efficacy in the inhibition of skin carcinogenesis.Support or Funding InformationWestern University of Health Sciences Intramural Grants