Combinative effects of β-elemene and propranolol on the proliferation, migration, and angiogenesis of hemangioma

Cell culture and treatment

HemECs were purchased from the Institute of Otwo Biotech (Shenzhen, China), and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured at 37 °C under 5% CO₂ and 95% air, HemECs were maintained in a DMEM medium supplemented with 10% FBS (Gibco, Billings, MT, USA) and 1% penicillin-streptomycin (FBS, Hyclone, Logan, UT, USA), HUVECs were maintained in ECM medium (Gibco, Billings, MT, USA). HemECs were exposed to either β-elemene (Dalian Holley Kingkong Pharmaceutical Co., Dalian, China) or propranolol (Sigma-Aldrich, Merck, St. Louis, MO, USA), or a combined treatment of β-elemene and propranolol for 24 h prior to analysis.

Calcein-AM/PI double staining

HemECs were incubated in six-well plates at a density of 5 × 10⁴ cells per well, Then the cells were treated with β-elemene or propranolol for 24 h. Next a Calcein-AM/PI Double Staining Kit (Cat No. MA0361; Meilun Biotech, Jiujiang, China) was used according to the manufacturer’s instructions, then incubated at 37 °C for 15 min. An excitation wavelength of 490 ± 10 nm was used to image living and dead cells using a fluorescence microscope (Olympus, Tokyo, Japan).

Cell counting Kit-8 (CCK-8) assay and LDH assays

Following the manufacturer’s instructions, cell proliferation was tested via a CCK-8 kit (Cat No. C0042; Beyotime Institute of Biotechnology, Jiangsu, China). 1 × 10⁴ cells per well were seeded into a 96-well plate for 24 h and treated with β-elemene or propranolol for 24 h, 48 h, and 72 h according to the study design. After the treatment, 10 µl of CCK-8 solution was added to each well and incubated for another 2 h at 37 °C. The optical cell density was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, we collected cell culture supernatants and used LDH activity kits (Cat No. C0017; Beyotime Institute of Biotechnology, Jiangsu, China) to measure lactate dehydrogenase (LDH) levels.

Scratch test

We plated 4 × 10⁴ HemECs per well for the scratch test in six-well plates. After the HemECs reached 100% confluency, the wounds were made using a yellow pipette tip. The HemECs were treated with β-elemene and propranolol and measured after 24 h. The experiment was repeated in triplicate. Data were expressed as the mean ± SD.

Tube formation assay

Matrigel Matrix (Corning, Corning, NY, USA) was added to each well of a 96-well plate and incubated at 37 °C for 30 min. Then we added HemECs (2 × 10⁴ cells/well) to 100 µL serum-free DMEM pipetted onto the Matrigel-coated plates and incubated at 37 °C for 8 h. The capillary-like structures were observed and imaged (Olympus, Toyko, Japan). The tube number was quantified using ImageJ software.

Western blot

Cells were harvested by centrifugation and lysed using RIPA lysis buffer (Cat No. G 2002-100ML; Servicebio, Wuhan, China) containing 1% protease inhibitor (Cat No. G2008-1ML; Servicebio, Wuhan, China) after treatment with β-elemene or propranolol for 24 h. Antibodies for HIF-1-α (Cat No. sc-13515; Santa Cruz Biotechnology, Dallas, TX, USA), VEGFA (Cat No. ab214424; Abcam, Cambridge, UK), phospho-Akt (Cat No. T56569; Abmart, Shanghai, China), Akt (Cat No. M63147; Abmart, Shanghai, China), phospho-Erk (Cat No. T57165; Abmart, Shanghai, China), Erk (Cat No. M63378, Abmart, Shanghai, China) were used in western blotting analyses. β-Tubulin (Cat No. M20005; Abmart, Shanghai, China) was used to confirm equal protein loading.

Total RNA extraction and real-time PCR

Total RNA was isolated with TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocol, and next the quantity and quality of RNA were assessed with Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), then approximately 1 microg of total RNA was reverse transcribed via HiScript III RT SuperMix (+gDNA wiper) (Vazyme, Nanjing, China). The qRT-PCR was carried out using a AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) with conditions following the manufacturer’s instructions. Relative gene expression levels were calculated using the 2^−ΔΔCt method. The primers used are as follows: HIF-1-α, 5′-AGCGACGTGGCTATTGTGAAG-3′(forward) and 5′-GCCATCATTCTTGAGGAGGAAGT-3′ (reverse); 18S, 5′-GAACGAGACTCTGGCATGCTA-3′ (forward) and 5′-CACGCTGAGCCAGTCAGTGTA-3′ (reverse); VEGFA, 5′-AGGGCAGAATCATCACGAAGT-3′ (forward) and 5′-AGGGTCTCGATTGGATGGCA-3′ (reverse); IL-1α, 5′-TGTATGTGACTGCCCAAGATG AAG-3′ (forward) and reverse: 5′-AGAGGAGGTTGGTCTCACTACC-3′ (reverse); IL-1 β, 5′-CCAGCTTCAAATCTCACAGCAG-3′ (forward) and reverse: 5′-CTTCTTTGGGTATTGCTTGGGATC-3′ (reverse); IL-6, 5′-CCTCCAGAACAGATTTGA GAGTAGT-3′ (forward) and 5′-GGGTCAGGGGTGGTTATTGC-3′ (reverse); IL-8, 5′-AAGACATACTCCAAACCTTTCCACC-3′ (forward) and 5′-CTTCAAAAACTTCTCCAC AACCCTC-3′ (reverse); IL-10, 5′-AACCTGCCTAACATGCTTCGA-3′ (forward) and 5′-CTCATGGCTTTGTAGATGCCT-3′ (reverse); TNF-α, 5′-CCCGCATCCCAGGACCTCTC T-3′ (forward) and 5′-CGGGGGACTGGCGA-3′ (reverse).

Xenograft experiments

Experiments were performed under a project license (No. SHCH-IACUC-2020-XMSB-36) granted by the Animal Ethics and Welfare Committee of Shanghai Children’s Hospital, in compliance with National Institutes of Health (NIH) Guide for the Care and Use of Animals. Five-week-old BALB/c male nude mice were purchased from Shanghai Laboratory Animal Center, China. The mice were housed in a specific-pathogen-free (SPF) environment with temperature set between 22 ± 2°C under 12 h light/dark cycle and relative humidity of 60 ± 10%. All the mice were given an unlimited supply of sterile food and water.

HemECs (5 × 10⁶) were inoculated into the flanks of the male BALB/c nude mice. Tumors were allowed to grow for 4 days. The initial tumor volume measurement was made using calipers. The mice were randomly divided into either the control, β-elemene, propranolol, or β-elemene + propranolol groups (four mice in each group). Mice were then treated daily with peritumoral injections of PBS (control group), β-elemene (75 mg/kg), propranolol (50 mg/kg), or β-elemene + propranolol. Tumors were measured with a caliper every 7 days, and the tumor volume was calculated using the formula V = length × width²/2. After 30 days of treatment, mice were euthanized using carbon dioxide, and the tumors were dissected and measured. The data are presented as mean ± SD.

Immunofluorescent staining

Tumors were embedded into Tissue-Tek optimum cutting temperature freezing media, cut into 5 µm cross sections, and fixed with 95% ethanol for 10 min. The sections were incubated with a CD31 primary antibody at 4 °C overnight after blocking. The tissue sections were then rinsed in phosphate buffered saline (PBS). The next day, the samples were incubated with a mixture of the corresponding secondary antibodies and imaged using an optical microscope. Microvessel density (MVD) was analyzed and measured using CD31 staining.

Statistical analysis

All results were repeated independently a minimum of three times and in triplicate. GraphPad Prism 5.0 was used for analysis. The differences among groups were measured using A two-tailed unpaired t-test and ANOVA. A p-value <0.05 was considered statistically significant.

Cytotoxic effect of propranolol and β-elemene on HemECs

The cytotoxic effects of β-elemene and propranolol were detected using an LDH assay. HemECs were incubated with different concentrations of β-elemene (25, 50, 75, 100, and 125 µg/ml) or propranolol (25, 50, 75, and 100 µM) for 24 h. The results showed no significant difference in the cytotoxic effect of β-elemene at concentrations lower than or equal to 75 µg/ml. Furthermore, there were no apparent cytotoxic effects of propranolol on HemECs at concentrations lower than or equal to 25 µM (Figs. 1A–1B). Cellular proliferation was measured via a CCK-8 method, as shown in Figs. 1C–1D. The proliferation of HemECs was inhibited at concentrations of β-elemene higher than or equal to 75 µg/ml, as well as at concentrations of propranolol that were higher than or equal to 50 µM. The results revealed that β-elemene was non-cytotoxic at the concentration of 75 µg/ml and produced inhibitory effects. Moreover, propranolol was cytotoxic at concentrations of 50 µM and produced inhibitory effects. To further study the cytotoxic effects of propranolol (50 µM, and 75 µM) and β-elemene (75 µg/ml), Calcein-AM/PI double staining was used. It was found that compared with the control, β-elemene had no apparent cytotoxic effects and propranolol at concentrations of 50 µM and 75µM was cytotoxic. Furthermore, by improving the propranolol concentration its cytotoxicity gradually increased. Combinatorial treatment using β-elemene and propranolol did not increase cytotoxicity compared to monotherapy with propranolol. The results suggest that combinatorial therapy with β-elemene and propranolol did not increase the cytotoxic effects of HemECs (Figs. 1E–1F). Therefore, 75 µg/ml β-elemene and 50 µM propranolol were used in subsequent experiments.

Combinatorial treatment using β-elemene and propranolol inhibited the migration, proliferation, and tube formation of HemECs without increasing cytotoxic side effects

Cellular migration was analyzed via a scratch test, cell proliferation was detected using the CCK-8 method, and tube formation was detected via a tube formation assay in order to investigate the combinatorial effects of β-elemene and propranolol on the migration and proliferation of HemECs. As shown in Fig. 2, compared with the single treatment group, combinatorial therapy using β-elemene (75 µg/ml) and propranolol (50 µM) significantly inhibited the migration, proliferation, and tube formation of HemECs. Additionally, the combinatorial treatment had the same inhibitory effects as the high-concentration propranolol treatment group (75 µM). Moreover, previous results showed that compared to the high-concentration propranolol group (75 µM), that combinatorial treatment with β-elemene (75 µg/ml) and propranolol (50 µM) had lower cytotoxicity. The results suggest that β-elemene combined with propranolol enhanced the inhibitory effect of HemECs without increasing the cytotoxic side effects.

HIF-1-α/VEGFA signaling pathway was activiated in hemangioma

To reveal how the HIF-1-α/VEGFA signaling pathway affects hemangioma proliferation, migration, and angiogenesis, We tested the expression of HIF-1-α, phospho-Akt, Akt, phospho-Erk, Erk, and VEGFA on protein levels in HUVECs and HemECs using Western Blot. The results showed that expression of HIF-1-α, phospho-Akt, phospho-Erk and VEGFA were higher than that of the HUVECs (Figs. 3B–3C). The RT-PCR results were consistent with that of the western blotting (Fig. 3A). Functional analysis confirmed that the ability of proliferation, migration, and tube formation in HemECs were more significant than that in the HUVECs (Figs. 3D–3H). Overall, our data demonstrated that HemECs compared with HUVECs, expressed higher levels of HIF-1-α and VEGFA, and exhibited a stronger growth potential. This conclusion helped us to further evaluate the effects of the combination of propranolol on HA.

Combinatorial treatment of β-elemene and propranolol inhibited the expression of HIF-1-α and VEGFA in HemECs

As previously reported, the HIF-1-α/VEGFA signaling pathway plays an important role during proliferation, migration, and angiogenesis. To evaluate the combinatorial treatment’s underlying mechanism, we divided the cells into specific groups; the control, β-elemene, propranolol, and combinatorial treatment groups and then exposed each group to hypoxia for 24 hours. Our study showed that compared with the control group, β-elemene and propranolol inhibited the expression of HIF-1-α, VEGFA, p-Akt/Akt, and p-Erk/Erk. However, the combinatorial treatment using β-elemene and propranolol produced significantly more inhibitory effects (Figs. 4C–4D). The RT-PCR results were consistent with that of the western blotting (Figs. 4A–4B), suggesting that combinatorial treatment synergistically down-regulates the HIF-1-α/VEGFA signaling pathway.

Combinatorial treatment of β-elemene and propranolol inhibited the cytokines in HemECs

It has been widely accepted that chronic inflammation increases tumorigenesis, development, and metastasis by creating an immunosuppressive microenvironment. As an inducer of tumors, inflammation could increase the risk of cancer by providing bioactive molecules, like cytokines, from cells infiltrating the tumor microenvironment. Therefore, inhibition of inflammation appears to be a promising therapy for anti-tumor treatment.

Our study showed that compared with the HUVECs, the expression of IL-6, IL-8 and TNF-α were increased in HemECs (Fig. 5A). We further evaluated the expression of cytokines in combinatorial treatment of β-elemene and propranolol. The data showed that the expression of cytokines were significantly inhibited in drug treatment groups compared with control, and combinatorial treatment of β-elemene and propranolol showed the better inhibitory effects than single treatment with β-elemene or propranolol (Fig. 5B).

Combinatorial treatment of β-elemene and propranolol inhibited the growth and angiogenesis of hemangioma

The suppressive effects of the combinatorial treatment of β-elemene and propranolol have been confirmed in HemECs. These effects were further validated in a mice xenograft tumor model. After 30 days of treatment using 75 mg/kg β-elemene and 50 mg/kg propranolol daily in nude mice, the tumor growth curve revealed that the tumor volume of the combinatorial treatment group was significantly smaller than that of the control, propranolol, and β-elemene monotherapy treatment groups (Figs. 6B–6C). Separated tumors of the HemEC cell xenografts and tumor weight showed consistent results (Fig. 6A and 6D). The CD31 antibody (red) was used as a microcapillary marker during immunofluorescence. As shown in Figs. 6E–6F, compared with the control, propranolol, or β-elemene monotherapy treatment groups, angiogenesis decreased significantly in the combinatorial treatment group. The results suggested that combinatorial treatment with β-elemene and propranolol significantly inhibited the growth and angiogenesis of hemangioma.