Propranolol, a non-selective β-blocker, is emerging as an effective treatment for complicated hemangiomas. The aim of this study was to investigate the molecular mechanism(s) underlying the therapeutic effects of propranolol against hemangiomas, using primary infantile hemangioma endothelial cells (IHECs). IHECs were treated with various concentrations of propranolol and morphological changes and apoptosis were assessed. Changes in the expression levels of apoptosis-related genes were examined. Annexin-V staining revealed that propranolol at 40, 50 and 60 μg/ml caused a concentration-dependent increase in the apoptosis of IHECs. Morphological analyses revealed that exposure to 50 μg/ml propranolol resulted in typical apoptotic changes, including shrinkage, the formation of apoptotic bodies and retention of plasma membrane integrity. Gene expression analyses revealed that propranolol treatment led to a marked increase in the expression of caspase-8, cytochrome c, apoptosis-inducing factor, caspase-3 and poly (ADP-ribose) polymerase 1, as well as a concomitant reduction in lamin B1 expression. Our data collectively demonstrate that propranolol induces apoptosis of IHECs through activation of the intrinsic and extrinsic apoptotic pathways, which represents an important mechanism for its therapeutic effects against infantile hemangiomas.

Abstract. Pingyangmycin (also known as Bleomycin A5) is produced by Streptomyces verticillus var. pingyangensis n.sp., and has anti-tumor activities against a variety of tumor cells. The aim of the present study was to determine the molecular mechanism(s) underlying the therapeutic effects of pingyangmycin against infantile hemangiomas. Human hemangioma-derived endothelial cells (HemECs) were treated with pingyangmycin at varying concentrations (100, 200 or 300 µg/ml), and the morphological changes and apoptosis levels were assessed. The gene expression changes were determined by cDNA microarray technology. Transmission electron microscopy examination revealed that the pingyangmycin-treated HemECs exhibited typical apoptotic characteristics, including chromatin condensation and the formation of apoptotic bodies. Annexin-V staining demonstrated that pingyangmycin caused a significant and dose-dependent induction of apoptosis in the HemECs. In the pingyangmycin-treated HemECs, 4,752 genes demonstrated at least 2-fold expression changes at the mRNA level. Quantitative polymerase chain reaction confirmed that pingyangmycin significantly upregulated the expression of p53, p53-induced protein with death domain, Bax, p53 upregulated modulator of apoptosis and p53 inducible gene 3, and downregulated the expression of murine double minute 2. The data demonstrated that the pro-apoptotic activity of pingyangmycin against infantile hemangiomas involves p53 pathway activation.

The use of propranolol for the treatment of infantile hemangioma (IH) has been widely investigated in recent years. However, the underlying therapeutic mechanism of propranolol for the treatment of IH remains poorly understood. The aim of the present study was to investigate the expression of proteins regulated by cellular tumor antigen p53 (p53) in associated apoptosis pathways in IH endothelial cells (HemECs) treated with propranolol. Furthermore, the present study aimed to investigate the exact apoptotic pathway underlying the therapeutic effect of propranolol against IH. In the present study, HemECs were subcultured and investigated using an inverted phase contrast microscope, immunocytochemical staining and a scanning electron microscope (SEM). Experimental groups and blank control groups were prepared. All groups were subjected to drug treatment. A high p53 expression model of HemECs was successfully established via transfection, and a low p53 expression model of HemECs was established using pifithrin-α. The apoptosis rate of each group was determined using Annexin V-fluorescein isothiocyanate/propidium iodide double staining and flow cytometry. The expression levels of downstream proteins regulated by p53 [tumour necrosis factor receptor superfamily member 6 (FAS), p53-induced death domain-containing protein (PIDD), death receptor 5 (DR5), BH3-interacting domain death agonist (BID), apoptosis regulator BAX (BAX), p53 unregulated modulator of apoptosis (PUMA), phosphatidylinositol-glycan biosynthesis class S protein (PIGS), and insulin-like growth factor-binding protein 3 (IGF-BP3)] were revealed in the experimental and control groups via western blotting. Microscopic observation revealed the growth of an adherent monolayer of cells, which were closely packed and exhibited contact inhibition. Immunocytochemical staining demonstrated increased expression of clotting factor VIII. SEM analysis revealed presence of Weibel-Palade bodies. The results of the analyses verified that the cultured cells were HemECs. The staining of the samples resulted in a significantly increased rate of apoptosis in experimental groups compared with the blank control group. This result suggested that there is an association between p53 expression and the rate of apoptosis of propranolol-treated HemECs. The results of the western blot analysis demonstrated an upregulation of BAX expression and a downregulation of IGF-BP3 expression in the HemECs treated with propranolol. There were no significant differences in the expression levels of FAS, DR5, PIDD, BID, PUMA and PIGS between experimental and control groups. This result suggests that p53 has an important role in HemEC apoptosis. The results of the present study additionally suggest that the propranolol-induced HemEC apoptosis pathway is a mitochondrial apoptosis pathway and is regulated by p53-BAX signaling.