Prevention of skin carcinogenesis by the β-blocker carvedilol

Abstract

The stress-related catecholamine hormones and the α- and β-adrenergic receptors (α- and β-AR) may affect carcinogenesis. The β-AR GRK/β-arrestin biased agonist carvedilol can induce β-AR-mediated transactivation of the EGFR. The initial purpose of this study was to determine whether carvedilol, through activation of EGFR, can promote cancer. Carvedilol failed to promote anchorage-independent growth of JB6 P(+) cells, a skin cell model used to study tumor promotion. However, at nontoxic concentrations, carvedilol dose dependently inhibited EGF-induced malignant transformation of JB6 P(+) cells, suggesting that carvedilol has chemopreventive activity against skin cancer. Such effect was not observed for the β-AR agonist isoproterenol and the β-AR antagonist atenolol. Gene expression, receptor binding, and functional studies indicate that JB6 P(+) cells only express β2-ARs. Carvedilol, but not atenolol, inhibited EGF-mediated activator protein-1 (AP-1) activation. A topical 7,12-dimethylbenz(α)anthracene (DMBA)-induced skin hyperplasia model in SENCAR mice was utilized to determine the in vivo cancer preventative activity of carvedilol. Both topical and oral carvedilol treatment inhibited DMBA-induced epidermal hyperplasia (P < 0.05) and reduced H-ras mutations; topical treatment being the most potent. However, in models of established cancer, carvedilol had modest to no inhibitory effect on tumor growth of human lung cancer A549 cells in vitro and in vivo. In conclusion, these results suggest that the cardiovascular drug carvedilol may be repurposed for skin cancer chemoprevention, but may not be an effective treatment of established tumors. More broadly, this study suggests that β-ARs may serve as a novel target for cancer prevention.

Figure 1. Effects of β-AR ligands on EGF-mediated neoplastic transformation of JB6 P+ cells

Effects of EGF or carvedilol (Car) on JB6 P+ cell transformation (A). The β-AR agonist isoproterenol (Iso) (B), the β-blockers carvedilol (C) and atenolol (Aten) (D) showed different degree of inhibition of EGF-induced cell transformation. Groups with different Greek letters are statistically different (p < 0.05) as determined by ANOVA with Tukey-Kramer post hoc test.

Figure 2. Comparison of the effects of carvedilol and atenolol on transformation and cytotoxicity of JB6 P+ cells

The β-blockers carvedilol (A) or atenolol (B) were examined for their effects on the cell transformation (by soft agar assay, in black) and cytotoxicity (by SRB assay, in gray) and data were normalized to their respective controls and plotted together. (C) Representative images of colonies after 10 days in soft agar within 12-well plates at 4x magnification; scale bar equals 1 mm.

Figure 3. β-adrenoceptor expression in JB6 P+ cells

(A) RT-PCR analysis revealed that JB6 P+ cells only express β2-ARs. The positive control (+Ctr) is Mouse Universal Reference cDNA. (B) Binding assays utilizing 3H-CGP and specific β-blockers: nebivolol (β1-AR), ICI-118,551 (β2-AR), and L-748,337 (β3-AR) indicate that only inhibition of the β2-AR showed significant (p < 0.05) displacement of CGP in a dose-dependent manner via an ANOVA with Tukey-Kramer post hoc test, indicated by an asterisk (*); n ≥ 6. (C) Functional assays examining cAMP accumulation utilizing specific agonists: xamoterol (β1-AR), formoterol (β2-AR), or L-755,507 (β3-AR) and carvedilol demonstrate that only stimulation of the β2-AR resulted in a statistically significant increase in cAMP compared to control (p < 0.05) via Kruskal-Wallis multiple-comparison test, indicated by an asterisk (*), which was reversed by carvedilol treatment.

Figure 4. Effects of carvedilol and atenolol on EGF-induced AP-1 transactivation

HEK-293 expressing an AP-1 luciferase reporter and renilla control were treated with vehicle control, EGF (10 ng/mL), EGF plus β-blockers carvedilol (A) or atenolol (B) for 24 h; data shown as means ± SE. Groups with different Greek letters are statistically different (p < 0.05) as determined by ANOVA with Tukey-Kramer post hoc test; n = 5.

Figure 5. Effects of carvedilol on DMBA-induced epidermal hyperplasia and the mutation of H-ras in SENCAR mice

(A) Representative microphotographs of the control and treated murine skin (H&E staining and PCNA expression). (B) Measurement of epidermal thickness; Groups with different Greek letters are statistically different (p < 0.05) as determined by ANOVA with Tukey-Kramer post hoc test. (C) H-ras CAA → CTA mutations from the epidermal samples shown in A and B. Groups with different Greek letters are statistically different (p < 0.05) as determined by ANOVA with Kruskal-Wallis Multiple-Comparison Z-Value with the Bonferroni Test; n ≥ 5.

Figure 6. Effects of carvedilol on human lung tumor A549 growth and migration in vitro and in vivo

(A) The β-blocker carvedilol was examined for effects on soft agar assay (in black) and SRB assay (in gray) and data were normalized to their respective controls and plotted together; asterisk (*) and cross (†) indicates the soft agar and SRB assay is statistically different than control, respectively, (p < 0.05) as determined by ANOVA with Tukey-Kramer post hoc test. (B) Cell migration of A549 cells was quantified by the gap distances before and after 24 hour treatment with carvedilol; asterisk (*) indicates that the 24 hour period was statistically different than the initial (p < 0.05) as determined by paired t-test, n = 3. (C) Effect of carvedilol on xenograft lung tumor growth in mice. Asterisk (*) indicates p-value is < 0.05 between the two points as determined via a RM-ANOVA with a Newman-Keuls multiple-comparison post hoc test; n ≥ 4.