Combined effect of propranolol, vincristine and bevacizumab on HUVECs and BJ cells

Abstract

Infantile hemangioma is one of the most common benign tumors affecting children, with ~10-15% requiring medical treatment. These tumors consist of endothelial cells and stromal components, including fibroblasts, pericytes and mast cells. Effects of propranolol treatment in combination with bevacizumab or vincristine on cell growth were compared in the current study using human umbilical vein endothelial cells (HUVECs) and BJ human normal fibroblasts (BJs) to determine potential synergic effects in vitro. Inhibition of cell growth was investigated using MTT assays and cytotoxicity of the drugs in various combinations was expressed as half inhibitory concentration (IC₅₀). Apoptosis was investigated using flow cytometry, with Alexa Fluor 488 and propidium iodide. Propranolol inhibited BJ and HUVEC growth in a dose-dependent manner, with increased response observed in BJs (IC50, 148,32 µg/ml; standard error logIC50, 0.07). Treatment with vincristine induced the strongest growth inhibition in HUVECs (IC50, 17,89 µg/ml; standard error log IC50, 0.07) and BJs (IC50, 24,81 µg/ml; standard error log IC50, 0.08) compared with propranolol (HUVEC IC50, 81,94 µg/ml; standard error log IC50, 0.06; BJ-IC50, 148,32 µg/ml; standard error logIC50, 0.07) or bevacizumab (HUVEC IC50 96,91 µg/ml; standard error log IC50, 0.06; BJ IC50, 182,70 µg/ml; standard error log IC50, 0.09) alone. Bevacizumab was the weakest cytotoxic agent. Combination treatment of vincristine with bevacizumab induced the highest levels of apoptosis in HUVECs compared with all other treatments and triple-drug therapy induced the levels of apoptosis in BJs. Single treatment with vincristine, propranolol or bevacizumab induced apoptosis in BJs and HUVECs. In BJs, triple treatment exhibited the greatest influence on apoptosis, compared with single and dual treatments and in HUVECs, vincristine and bevacizumab combination treatment induced apoptosis to the highest level. The present study offers novel perspectives in drug repurposing studies for the three drugs, particularly in diseases where the pathogenesis is based on healthy endothelial cell proliferation, including hemangiomas.

Keywords: bevacizumab; fibroblasts; human umbilical vein endothelial cells; infantile hemangioma; propranolol; vincristine.

Figure 1.

Inhibitory effect of propranolol, vincristine and bevacizumab in HUVECs and BJs. Cell viability determined by MTT assay is presented as a function propranolol in (A) HUVECs and (B) BJs, vincristine in (C) HUVECs and (D) BJs, and bevacizumab in (E) HUVECs and (F) BJs. *P<0.05; **P<0.01; and ***P<0.001 vs. untreated (0 µg/ml). HUVEC, human umbilical vein endothelial cell; BJ, CRL-2522 human normal fibroblast.

Figure 2.

Inhibitory effect of propranolol plus vincristine, propranolol plus bevacizumab and vincristine plus bevacizumab in HUVECs and BJs. Cell viability determined by MTT assay is presented as a function of drug concentration, with varying propranolol plus 10 µg/ml vincristine in (A) HUVECs and (B) BJs, with varying propranolol plus 100 mg/ml bevacizumab in (C) HUVECs and (D) BJs, and with varying vincristine plus 100 mg/ml bevacizumab in (E) HUVECs and (F) BJs. *P<0.05; **P<0.01; and ***P<0.001 vs. untreated (0 µg/ml). HUVEC, human umbilical vein endothelial cell; BJ, CRL-2522 human normal fibroblast.

Figure 3.

Apoptosis evaluation of HUVECs treated with propranolol, vincristine and bevacizumab and combinations of the drugs. Representative images of flow cytometry-based apoptosis evaluation of HUVECs (A) untreated or treated with (B) propranolol (50 µg/ml), (C) vincristine (10 µg/ml), (D) bevacizumab (50 µg/ml), (E) propranolol (50 µg/ml) plus vincristine (10 µg/ml), (F) propranolol (50 µg/ml) plus bevacizumab (50 µg/ml), (G) vincristine (10 µg/ml) plus bevacizumab (50 µg/ml), and (H) propranolol (50 µg/ml) plus vincristine (10 µg/ml) plus bevacizumab (50 µg/ml). HUVECs, human umbilical vein endothelial cell; Q1, quadrant representative of early apoptosis; Q2, quadrant representative of late apoptosis; Q3, quadrant representative of viable cells; Q4, quadrant representative of dead cells.

Figure 4.

Apoptosis evaluation of BJs treated with propranolol, vincristine and bevacizumab and combinations of the drugs. Representative images of flow cytometry-based apoptosis evaluation of BJs (A) untreated or treated with (B) propranolol (50 µg/ml), (C) vincristine (10 µg/ml), (D) bevacizumab (50 µg/ml), (E) propranolol (50 µg/ml) plus vincristine (10 µg/ml), (F) propranolol (50 µg/ml) plus bevacizumab (50 µg/ml), (G) vincristine (10 µg/ml) plus bevacizumab (50 µg/ml), and (H) propranolol (50 µg/ml) plus vincristine (10 µg/ml) plus bevacizumab (50 µg/ml). BJ, CRL-2522 human normal fibroblast; Q1, quadrant representative of early apoptosis; Q2, quadrant representative of late apoptosis; Q3, quadrant representative of viable cells; Q4, quadrant representative of dead cells.

Figure 5.

Quantitative evaluation of apoptosis in HUVECs and BJs treated with propranolol, vincristine and bevacizumab and combinations of the drugs. Number early and late apoptotic and dead cells determined by flow cytometry using (A) HUVECs and (B) BJs treated with propranolol (50 µg/ml), vincristine (10 µg/ml) and bevacizumab (50 µg/ml) and combinations of the drugs. Values are per population of 10,000 cells. HUVEC, human umbilical vein endothelial cell; BJ, CRL-2522 human normal fibroblast; P, propranolol; V, vincristine; B, bevacizumab.