Propranolol exhibits activity against hemangiomas independent of beta blockade

Abstract

Propranolol is a widely used beta blocker that consists of a racemic mixture of R and S stereoisomers. Only the S stereoisomer has significant activity against the beta-adrenergic receptor. A fortuitous clinical observation was made in an infant who received propranolol for cardiac disease, and regression of a hemangioma of infancy was noted. This has led to the widespread use of propranolol for the treatment of large and life-threatening hemangiomas of infancy. Infants receiving propranolol require monitoring to ensure that they do not suffer from side effects related to beta blockade. The exact mechanism of activity of propranolol in hemangioma of infancy is unknown. In this study, we treated hemangioma stem cells with both beta blockade active S- and inactive R-propranolol and looked for genes that were coordinately regulated by this treatment. Among the genes commonly downregulated, Angiopoietin-like 4 (ANGPTL4) was among the most regulated. We confirmed that propranolol isomers downregulated ANGPTL4 in endothelial cells, with greater downregulation of ANGPTL4 using the beta blockade inactive R-propranolol. ANGPTL4 is present in human hemangiomas of infancy. Finally, R-propranolol inhibited the growth of bEnd.3 hemangioma cells in vivo. The implication of this is that hemangioma growth can be blocked without the side effects of beta blockade. Given that humans have been exposed to racemic propranolol for decades and thus to R-propranolol, clinical development of R-propranolol for hemangiomas of infancy and other angiogenic diseases is warranted.

Keywords: Paediatric cancer; Tumour angiogenesis.

Fig. 1

Hierarchical clustering of differentially expressed genes of HemSCs treated with R-propranolol. a Heat map of genes that are upregulated in R-propranolol-treated HemSCs. b Heat map of genes that are downregulated in R-propranolol-treated HemSC

Fig. 2

List of 100 most upregulated (red) and downregulated (blue) genes in HemSCs. List of genes upregulated or downregulated in R- or S-propranolol-treated HemSC samples when compared to the vehicle control was sorted by the significance (p < 0.05). ANGPTL4 is the most downregulated gene in R-propranolol-treated HemSCs

Fig. 3

ANGPTL4 protein expression is reduced in R-propranolol-treated bEnd.3 cells. bEnd.3 cells were seeded 24 h prior to the R- or S-propranolol treatment. Culturing media were changed to complete media containing either 10 μM R- or S-propranolol or vehicle control of ethanol. After 24 h propranolol treatment, cells were subjected to western blotting and probed for ANGPTL4. R-propranolol differentially reduced the expression of ANGPTL4 as demonstrated by detection of the 55 kDa expected size bands. Loading control of GAPDH demonstrates that reduction is not due to the loading. Three independent experiments were performed, and a representative image is shown here. Other experimental results from western blotting is described in Supplementary Fig. 2. Titration of R-propranolol ranging from 0 to 10 μM was performed to determine the optimal treatment concentration and described in Supplementary Fig. 3

Fig. 4

ANGPTL4 is expressed in hemangioma of infancy. Representative images of immunohistochemistry. The panels represent (a) no antibody control, (b) human infant foreskin sample stained with ANGPTL4, and (c) infantile hemangioma sample stained with Angptl4. The scale bars represent 100 μm (a, c) and 50 μm (b). Infantile hemangioma samples highly express ANGPTL4 proteins in endothelial cells, while human foreskin sample consists of largely negative cells surrounded by artifactually stained tissue

Fig. 5

R-propranolol significantly suppressed tumor growth in vivo. bEnd.3 cells allografted into mice formed tumors within 2 weeks of inoculation, and treatment with R-propranolol significantly reduced the tumor volume within 2 weeks of tumor development (p = 0.014). Five animals were included in each of the control and the treatment groups, depicted by individual dot in the graph. All statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA)

Fig. 6

Heat map of differentially expressed RNA between bEnd.3 cells treated with R-propranolol and vehicle. Twenty-four transcripts were identified by RNAseq to be upregulated or downregulated in R-propranolol-treated cells when compared to the vehicle-treated control cells with p value ≤ 0.05. Transcripts were further filtered by minimum two-fold difference in the expression levels

Fig. 7

Selection of box plots of differentially expressed RNA transcripts of bEnd.3 cells treated with R-propranolol. Seven transcripts were found to be significantly downregulated, and 17 transcripts were upregulated. Six of the upregulated genes of interest including Egr1, APOA1, and BHMT as well as three of the downregulated genes including Faim2, Hunk, and Eno4 are included for representation. Box plots represent interquartile range with the central line denoting the median, and upper and lower whiskers represent standard error of means. Individual data points are included to demonstrate the spread

Fig. 8

R-propranolol alters changes in the expression of proteins identified in RNAseq, validating the findings. Immunohistochemistry for ANGPTL4, BHMT, and APOA1 were performed on paraffin-embedded samples of R-propranolol- or ethanol vehicle-treated bEnd.3 murine tumor to validate the differential expression analysis results obtained using RNAseq. Nuclear expression of ANGPTL4 was markedly reduced in R-propranolol-treated animals while BHMT and APOA1 expression was increased in the experimental group, supporting the RNAseq findings. a, b Control- and R-propranolol-treated tumor samples stained with ANGPTL4. c, d Control- and R-propranolol-treated tumor samples stained with BHMT. e, f Control- and R-propranolol-treated tumor samples stained with APOA1. Scale bars indicate 50 μm in all panels

Fig. 9

Proposed mechanism of action of propranolol in hemangioma of infancy. Proliferative hemangiomas are glycolytic and have elevated levels of VEGF, Ang2, and ANGPTL4, resulting in a highly angiogenic tumor with leaky vessels. Treatment with propranolol results in conversion to a respiratory phenotype, with downregulation of VEGF and ANGPTL4, but retaining Ang2, which then mediates regression of hemangiomas in the absence of angiogenic stimulation