Oridonin exhibits anti-angiogenic activity in human umbilical vein endothelial cells by inhibiting VEGF-induced VEGFR-2 signaling pathway

Jin-Huan Jiang (Conceptualization) (Methodology) (Software) (Data curation) (Investigation) (Formal analysis) (Writing – original draft), Jiang Pi (Methodology) (Software) (Formal analysis), Ji-Ye Cai (Conceptualization) (Validation) (Writing – review and editing) (Resources) (Visualization)

Abstracts

Oridonin has been found to be a potential anti-angiogenesis agent. However, its functional targets and the underlying mechanisms are still vague. In vitro studies we found that oridonin not only inhibited VEGF-induced cell proliferation, migration and tube formation but also caused G2/M phase arrest and triggered cellular apoptosis in HUVECs. In mechanistic studies revealed that oridonin exhibited the anti-angiogenic potency, at least in part, through the down-regulation of VEGFR2-mediated FAK/MMPs, mTOR/PI3K/Akt and ERK/p38 signaling pathways which led to reduced invasion, migration, and tube formation in HUVECs. Our results could provide evidence that oridonin exerts strong anti-angiogenesis activities via specifically targeting VEGFR2 and its signaling pathway.

Key words: Oridonin, Anti-angiogenesis, VEGF, VEGFR2, HUVECs

1. Introduction

The growth and spread of solid tumors require concomitant expansion of vascular networks to transport oxygen and other essential nutrients. Hence, the gradual development of tumors is able to induce neovascularization by a process called angiogenesis[1, 2]. So, angiogenesis plays a vital role in the growth and spread of cancer as an adequate blood supply, which is necessary for tumor growth and invasion in normal tissues[3]. These findings lead to a promising strategy for cancer therapy by inhibiting the neovascularization to prevent the cancer cells growth and metastasis. Indeed, anti-angiogenesis presents a very preferential goal for cancer treatment, and researchers have already made sizable efforts to discover new natural and synthetic anti-angiogenic agents[4-6]. On the basis of carcinogenic stimulation, elevated metabolic activity and mitochondrial dysfunction, tumor cells could develop their microenvironment via producing a high level of pro-angiogenic factor VEGF[7, 8]. As one of the most efficient regulators of angiogenesis, VEGF exerts crucial roles in the endothelial cell survival, proliferation, migration, invasion, and capillary-like tube formation[9-11]. The biological actions of VEGF are mediated by three high-affinity trans-membrane receptor tyrosine kinases named VEGFR1, VEGFR2 and VEGFR3[9, 12]. Among these three receptors, VEGFR2 has emerged as the primary receptor of VEGF and the major effector of VEGF-induced pro-angiogenesis signaling in endothelial cells[13, 14]. When VEGF binds to VEGFR2 on endothelial cells, several key intracellular signaling molecules, such as focal adhesion kinase (FAK), secretion of matrix metalloproteinases (MMPs) can be activated, which are responsible for endothelial cell migration, proliferation, and tubulogenesis[15, 16].

Additionally, VEGFR2 signaling activation could cause the phosphorylation of some downstream signal transduction mediators, including PI3K/AKT/mTOR, and MAPK, which also ultimately control processes critical to angiogenesis[17]. Therefore, VEGFR2 is deemed as potential molecular target for anti-angiogenic against cancer metastasis. Studies have shown that inhibition of endothelial cells growth can block tumor angiogenesis effectively[18, 19]. Several natural products have been well documented to reduce VEGF-activated angiogenesis via impeding the activation of VEGFR2 signaling pathways[16, 17, 20, 21]. Therefore, inhibitors of VEGF and its receptors (VEGFR2) with anti-angiogenic activities are widely pursued as hopeful candidates for solid tumor treatment. As an effective diterpenoid, oridonin has been shown to own potent antitumor activity[22-24].According to a few reports, oridonin can restrain the formation of capillary-like networks, manifesting that it has anti-angiogenic activity[25]. Dong et al. certified that oridonin could slow the colon tumor growth and metastasis mainly via prohibiting tumor angiogenesis by interfering with the VEGF-stimulated Jagged-Notch signaling pathway[26]. For the in vivo study, Tian et al. confirmed that oridonin appeared attractive anti-angiogenesis effects attributing to a lower expression of VEGFR2, VEGFR3 and VEGFA in zebra fish model[25]. Nevertheless, the anti-tumor mechanism of oridonin, especially it’s potential anti-angiogenesis mechanisms, are still largely unknown. So, in this part, we have depicted the role of oridonin in angiogenesis and characterized the underlying molecular mechanisms.

2. Materials and methods

2.1 Materials

Fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM) (Gibco, Big Cabin, Oklahoma, ME, USA). Oridonin (>98%, HPLC) was procured from Ming wang Biotechology (GuangZhou, China). 3-(4, 5)-dimethylthiazo(-z-y1)-3,5-diphenyt-etrazoliumromide (MTT), Vascular endothelial growth factor (VEGF) were purchased from Sigma (Saint Louis, USA). Rhodamine-123 was obtained from Beyotime Institute of Biotechnology (Shanghai, China). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), JC-1 mitochondrial potential sensors were purchased from Invitrogen Molecular Probes
(Indiana, USA). VEGFR2, p-VEGFR2, FAK, p-FAK, MMP-2, MMP-9, m-TOR, PI3K, Akt, ERK, p38, p-m-TOR, p-ERK, p-PI3K, p-Akt, p-p38, GAPDH, β-actin
antibodies were obtained from Cell Signaling Technology (1:1000, Danvers, USA). Fluorescent secondary antibodies (anti-rabbit/mouse IgG, 1:5000) were procured from Santa Cruz Biotechnology (Santa Cruz, USA). Matrigel matrix, Annexin V-FITC/PI apoptosis detection and cycle analysis kits were purchased from BD Biosciences (Bedford, USA).

2.2 Cell culture

Primary human umbilical vein endothelial cells (HUVECs) were obtained from College of Life Science and Technology Jinan University (Guangzhou, China). Cells were cultured in DMEM medium containing 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2.All cells were pass aged every three days.

2.3 Cell viability assay

HUVECs cells (5*103 cells /100 μl) were seeded into 96 well plates and treated with a range of concentrations of oridonin (0, 2.5, 5, 10, 20 μM) for 24 h and 48 h, respectively. The effects of oridonin on HUVECs viability under VEGF-stimulated condition, HUVECs (5*103 cells/well) were respectively treated with different concentrations of oridonin (2.5, 5, 10, 20 μM) in the absence or presence of VEGF (50 ng/ml) for 24 h and 48 h, respectively. In all experiments, cell viability was determined by MTT assay. MTT reagent (10 μl) was added into each well for 4 h incubation. The absorbance was measured at 570 nm by a spectrophotometer (Tecan, Switzer-land).

2.4 Wound healing migration assay

To evaluate the effects of oridonin on cell migration, a scratch assay was applied. HUVECs were seeded at a density of 2~4*105 cells per well in a 6-well culture plate, and cultured for one day in DMEM containing 10% FBS. It can be covered by overnight. Later, using a 10 µl pipette tip perpendicular to the cell surface to scratch a straight line. The tip should be vertical and not tilted. Then, aspirating old DMEM medium and using PBS wash cell surface 3 times to remove the scratched cells. Following, DMEM supplemented with 0.5% FBS was added into the each wells with or without VEGF (50 ng/ml) and different dose of oridonin (10, 20 μM). Then, HUVECs were allowed to migrate for 24 h at 37 °C in 5% CO2 and the healing processes were captured by an inverted microscope with ×100 magnification. The migrated cells were quantified by manual counting. Inhibition percentage was expressed as percentage of the vehicle control (100%).

2.5 Tube formation assay

The effect of oridonin on angiogenesis in vitro was evaluated by the tube formation assay. Advance preparation: firstly, thaw an appropriate volume of matrigel in a 4 °C refrigerator one day in advance. Secondly, pre-cool the 200 µl pipette tips and 48-well plate in a -20 °C refrigerator one day in advance to prevent 4 °C from solidifying during use. Experimental procedure: the pre-cool 48-well plate was coated with 50µl matrigel per well, gently shaking the plate until the matrigel was evenly distributed over the whole well, and be careful not to create bubbles, and allowed it to solidify at 37 ˚C for 1 h. Then, HUVECs were placed at a density about 2~3*104 cells/well into the pre-cool 48-well plate, and incubated in DMEM supplemented with 0.5% FBS for 6 h. Later, all cells were treated with or without VEGF (50 ng/ml) and followed by the addition of oridonin (10, 20 μM) for another 6 h exposure. HUVECs tube or network-like structures were imaged under a light microscope with ×200 magnification.

2.6 Intracellular F-actin imaging and immunofluorescence analysis

HUVECs (2.5*104 /ml) were seeded into confocal plates for 24 h. Cells were stimulated with or without 50 ng/ml VEGF for 30 min, and then oridonin ( 10, 20 μM) treated for another 12 h. Later, the cells were fixed in 4% PFA for 10 min at room temperature and followed by 0.1% Triton X-100 permeabilized treatment. After washing three times with PBS, F-actin filaments were stained with 1 μM rhodamine-phalloidin for 1 h and nuclei were followed by 50 μM DAPI for 5 min. Next, all cells were washed with PBS three times again. At last, a confocal microscopy (Leica TCS SP2, Wetzlar, Germany) was applied for imaging the cell nucleus and the organization of F-actin cytoskeleton structure.

2.7 Cell cycle detection
HUVECs cells (1*105 cells /2ml) were seeded into 6 well plates, cells were treated with or without VEGF (50 ng/ml) for 30 min, then oridonin (10, 20 μM) incubated for another 24 h. Later, all cells were harvested and fixed with 70% ethanol overnight at 4°C. The fixed cells were washed with PBS, treated with RNase A/ PI for 30 min at 37 °C. Cell cycle phase distribution was detected by flow cytometer (BD Biosciences, Bedford, USA).

2.8 Cell apoptosis detection

All cells were incubated with oridonin or VEGF in accordance with for cell cycle detection. Annexin V-FITC/PI apoptosis detection kit was used to detect the cell apoptosis. Cells were harvested and stained with the FITC-labeled Annexin V and PI for 15 min under dark conditions. Further procedure for the population of apoptosis was evaluated immediately by flow cytometer .

2.9 Mitochondrial membrane potential (