Beta blockade synergizes with chemotherapy to induce apoptosis of malignant vascular tumor cells and does not result in accumulation of chemoresistance.

<p>(<b>A</b>) The panel of malignant vascular tumor cells were treated with 100 µM propranolol, cisplatin (cis), busulfan (bus), vincristine (vin), or H₂O₂ alone or in combination and cell survival was assessed after 24 hours. (<b>B</b>) The cell panel was treated with 100 µM propranolol until approximately 5% of the culture remained alive. The remaining cells were allowed to repopulate the culture and this process was repeated three subsequent times. Cell survival was assessed after 24 hours of 100 µM propranolol treatment of the naïve (1^st) tumor cells and selected (4^th) tumor cells. For all experiments, the data is the average +/− standard deviation for at least three biological replicates. Statistical significant was determined using Students t-test (p<0.05).</p

Beta blockade induces alterations in key cell cycle regulators and the Tie2 angiogenic regulator.

<p>(<b>A</b>) qPCR analysis of a panel of 84 genes involved in cell cycle regulation. Shown are the 25 genes whose mRNA expression levels were statistically altered 1.5 fold or more following 24 hours of 100 µM propranolol in SVR cells. The qPCR data is the average +/− standard deviation for at least three biological replicates. Statistical significant was determined using Students t-test (p<0.05). (<b>B</b>) Western blot detecting cell cycle regulatory protein levels in SVR cells subjected to 24 hours of sham or 100 µM propranolol. (<b>C</b>) Immunofluorescent staining for p21 (<i>red</i>) and p27 (<i>green</i>) in SVR cells subjected to 24 hours of sham or 100 µM propranolol. (<b>D</b>) SVR cells were grown in standard growth media (10% FBS) or serum starved overnight, treated as indicated with sham, 100 µM propranolol (24 hours), or 10 ng/ml angiopoietin-1 (2.5 minutes), and Western analysis detected the expression of phosphorylated Tie2, total Tie2, and actin.</p

Beta blockade induces apoptosis of malignant vascular tumor cells.

<p>(<b>A</b>) Apoptosis quantification of Hoechst 33342 and propidium iodide stained malignant vascular tumor cells after 24 hours treatment with sham or 100 µM propranolol. (<b>B</b>) Fluorescent images of SVR angiosarcoma cells stained with Hoechst 33342 and propidium iodide after 24 hours treatment with sham or 100 µM propranolol. (<b>C</b>) 3D rendering of representative SVR angiosarcoma cell nuclei after 24 hours treatment with sham or 100 µM propranolol. (<b>D</b>) qPCR analysis of a panel of 84 genes involved in apoptotic regulation. Shown are the 18 genes whose steady state mRNA expression levels were statistically altered 1.5 fold or more following 24 hours of 100 µM propranolol in SVR cells. (<b>E</b>) Immunofluorescent detection of cleaved caspase-3 in SVR angiosarcoma cells after 24 hours treatment with sham or 100 µM propranolol. (<b>F</b>) Western analysis detecting phospho-p38 MAPK and total p38 MAPK protein levels after 1 hour of sham or 100 µM propranolol treatment of SVR angiosarcoma cells. (<b>G</b>) Western blot detection of apoptotic protein levels. (<b>H</b>) Unstained SVR angiosarcoma cells were co-cultured with CellTracker Blue stained HDMVECs. The co-cultures were subjected to sham or 100 µM propranolol and DIC/fluorescent image overlays were obtained after 48 hours treatment. For all experiments, the data is the average +/− standard deviation for at least three biological replicates. Statistical significant was determined using Students t-test (p<0.05).</p

Clinical data for angiosarcoma tissues.

<p>Clinical data for angiosarcoma tissues.</p

Beta blockade inhibits malignant vascular tumor cell proliferation in a dose dependent manner.

<p>(<b>A</b>) The panel of malignant vascular tumor lines was subjected to a dose curve of propranolol over a 48 hour time course and changes in cell proliferation were quantified by counting the number of cells per vision field. (<b>B</b>) Representative images of sham or 100 µM propranolol treated SVR angiosarcoma cells after 48 hours. (<b>C</b>) The panel of malignant vascular tumor lines was grown on Alvetex polystyrene membranes for 48 hours, treated with sham or 25 µM propranolol, and cell density was accessed by quantifying positive Hoechst nuclear staining after 96 hours of treatment. (<b>D</b>) Differential interference contast (DIC) and fluorescence image overlays of sham or 25 µM propranolol treated SVR angiosarcoma cells in the Alvetex membranes 96 hours after treatment. (<b>E</b>) Cell cycle analysis of propidium iodide stained SVR angiosarcoma cells treated with sham or 100 µM propranolol for 24 hours. (<b>F</b>) Cell cycle profile quantification of malignant vascular tumor cells treated with sham or 100 µM propranolol for 24 hours. For all experiments, the data is the average +/− standard deviation for at least three biological replicates. Statistical significant was determined using Students t-test (p<0.05).</p

Three fold or greater changes in gene expression (p<0.05) after 24 hours of 100 mM propranolol in SVR angiosarcoma cells.

<p>Three fold or greater changes in gene expression (p<0.05) after 24 hours of 100 mM propranolol in SVR angiosarcoma cells.</p

Beta adrenergic receptor expression in malignant vascular tumors.

<p>(<b>A</b>) Alkaline phosphatase detection of ADRB1, ADRB2, and ADRB3 protein (<i>red</i>) in H&E stained human angiosarcoma paraffin tissue sections. (<b>B & C</b>) qPCR detection of ADRB1, ADRB2, and ADRB3 mRNA in a panel of canine (<b>B</b>) and mouse (<b>C</b>) malignant vascular tumor lines Data is provided as the average gene expression +/− standard deviation of at least triplicate biological replicates for each gene.</p