Abstract
Purpose: To study the inhibitory effect of different doses of tetramethylpyrazine (TMP) combined with the beta-blocker, propranolol (Pro) on hemangioma endothelial (EOMA) cells.
 Methods: EOMA cells were cultured in vitro with varying doses of TMP and Pro (5, 10, 20 and 40 uM). The effect of treatments on cell proliferation was assessed by MTT assay, while cell apoptosis was assayed by flow cytometry. The expressions of Bcl-2, Bax, p-mTOR), total-mammalian target of rapamycin (t-mTOR, p-p70S6) and total-p70 ribosomal protein S6 (t-p70S6) proteins were determined using Western blot.
 Results: MTT data showed that when used alone, TMP had no significant inhibitory effect on EOMA cells (p > 0.05). However, when TMP was combined with propranolol, there was significant inhibition of EOMA cells, and that the inhibition is dependent on TMP dose. Flow cytometry results showed that the combination of TMP and Pro induced EOMA cell apoptosis dose-dependently (p < 0.05). Moreover, TMP dose-dependently inhibited the phosphorylation of mTOR and p70S6 in EOMA cells, and enhanced Bax expression, but downregulated Bcl-2 (p < 0.05).
 Conclusion: These results suggest that TMP enhances the inhibitory influence of Pro p-mTOR and pp-70S6 in EOMA cells in a dose-dependent manner. Thus, TMP may enhance Pro-induced inhibition of the growth of endothelial cells, and promote apoptosis through suppression of activation of PI3K/AKT signal route. These findings provide a theoretical basis for the clinical application of TMP/Pro combination for the treatment of hemangioma.
Highlights
Hemangioma is a congenital benign tumor or vascular malformation that often occurs in the skin and soft tissues of infants and young children
The EOMA cells were seeded at a density of 1 × 109 cells/L in a 6-well culture plate with medium (1.2 mL per well)
Influence of combination of TMP and Pro on the expressions of Bcl-2, Bax, P-mTOR, TmTOR, P-p70S6 and T-p70S6 proteins in EOMA cells Western blot assay showed that treatment of hemangioma endothelial cells with combination of TMP and Pro, decreased P-Mtor/T-mTOR and P-p70S6/T-p70S6 ratios
Summary
Hemangioma is a congenital benign tumor or vascular malformation that often occurs in the skin and soft tissues of infants and young children. There are limited reports on the effect of TMP on hemangioma endothelial cells in China and elsewhere. The effect of combination of TMP and Pro on EOMA cells, and the possible mechanism involved were investigated in this study. A blank control group was set up with culture medium but without cells. Mouse hemangioma EOMA cells were supplied by American Type Culture Collection (ATCC); Low-sugar DMEM was added (200 μL per well), and was aspirated after culturing for 24 h, followed by drug addition. Numeric data are expressed as mean ± SD, and comparison between groups was performed using one-way analysis of variance. Values of p < 0.05 were considered statistically significant
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