Anti-angiogenic and antitumor activities of Huaier aqueous extract

Abstract

Traditional Chinese medicine, a rich source of potent cancer chemopreventive agents, is attracting increasing attention worldwide. Recently, the anticancer activity of Trametes robiniophila Μurr. (Huaier) has been widely investigated. However, the mechanisms are not yet fully understood. This study aimed to elucidate the inhibitory effect of Huaier extract on angiogenesis and tumor growth. Incubation with Huaier extract inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and mouse mammary tumor cells (4T1). In addition, treatment with Huaier extract decreased the motility and tube formation of HUVECs in a dose-dependent manner in vitro. As determined by western blot analysis, Huaier extract dose-dependently decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK), transcription factor p65, c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription 3 (STAT3) and the expression of vascular endothelial growth factor (VEGF). In ex vivo experiments, new vessel growth was suppressed as shown by chick embryo chorioallantoic membrane (CAM) and rat aortic ring assays in the presence of Huaier extract. To further evaluate the inhibitory effect, 4T1 cells were injected subcutaneously into BALB/c mice. The administration of Huaier extract suppressed tumor volume, decreased microvessel density and induced apoptosis. These data suggest that Huaier extract may serve as a potent anti-angiogenic and antitumor agent.

Figures

Figure 1

Morphological and viability changes in HUVECs induced by Huaier extract. (A) HUVECs were seeded in 24-well plates and incubated with Huaier extract for 24 h. Representative images of HUVECs in the control and treated groups. (B) Huaier extract inhibited cell proliferation in a time- and dose-dependent manner. The results are presented as the means ± SD of 3 independent experiments conducted in triplicate. *P<0.05; **P<0.01; #P<0.05; ##P<0.01.

Figure 2

Huaier extract induced apoptosis and caused cell cycle arrest in HUVECs. HUVECs were incubated with increasing concentrations of Huaier extract for up to 48 h. (A and C) Examples of flow cytometric data of HUVECs examined by PI-Annexin-V staining analysis after incubation with Huaier extract for 48 h. (B and D) Representative images of cell cycle analysis are shown, and the percentage of cells in each phase of the cell of each following treatment for 24 h is presented as a histogram, and all values were expressed as the means ± SD of 3 independent experiments conducted in triplicate. (E and F) Huaier extract induced the accumulation of p21. The bar graphs represent the relative density of p21/actin. Con, control. *P<0.05; **P<0.01; #P<0.05; ##P<0.01.

Figure 3

Effect of Huaier extract on the cell migration of HUVECs. (A) Confluent monolayers of HUVECs on a 12-well plate were wounded using a pipette tip and treated with Huaier extract or the vehicle. The images of wound closure were captured under a phase-contrast microscope after 48 h. (B) The migration inhibition is presented as distances between the 2 edges of the scratch. (C) Chemotaxis assay of HUVECs, which shows the inhibitory effect of Huaier extract on cell migration following 24 h of treatment. The cells successfully migrated to the lower surface of the insert. (D) The cell numbers decreased in a dose-dependent manner following treatment with Huaier extract. In the histograms, all values are expressed as the means ± SD. *P<0.05; **P<0.01 as compared with the vehicle.

Figure 4

Huaier inhibited angiogenesis in vitro and ex vivo. (A) HUVECs were seeded on Matrigel-coated 96-well plates and incubated in the absence or presence of Huaier extract for 9 h. Representative images of HUVEC tube formation. (B) CAM of 9-day-old chick embryos exposed to Huaier extract or the vehicle. After 24 h of incubation, the CAM tissue directly beneath each filter disc was photographed. The image represents at least 6 chick embryos. (C) Examples of aortic rings of mice fed with DMEM containing 20% FBS in the control and treated groups. Representative images of vessel sprouting were taken at day 5 of treatment.

Figure 5

Huaier extract regulated multiple molecules in HUVECs. (A) After HUVECs were treated with various concentrations of Huaier extract, the expression levels of phosphorylated (P)-ERK and ERK were detected after 2 h, while the expression of VEGF was analyzed after 24 h by immunoblotting. β-actin was used as the loading control. (B) The bar graphs represent the mean relative densities ± SD of 3 independent experiments. Con, control. *P<0.05; **P<0.01.

Figure 6

Huaier extract suppressed the growth of 4T1 cells in vitro and in vivo. (A) Huaier extract inhibited 4T1 cell proliferation as shown by MTT assay. In the in vivo experiment, the mice were administrated with Huaier extract by gavage daily for 18 days. (B) Tumor images after mice were sacrificed. (C) Inhibition of tumor growth judging from the final tumor weight. (D and E) Tumor volumes and body weights were measured 7 days later. The bars represent the means ± SD. *P<0.05; **P<0.01; #P<0.05; ##P<0.01.

Figure 7

H&E, CD34 and TUNEL staining of tumor tissues. 4T1 xenograft tissues were removed and cut into 4-μm thick slices. Representative images of (A) H&E staining showing the necrotic areas, (B) CD34 stainig, as an indicator of tumor microvessel density, and (C) TUNEL staining, where TUNEL-positive cells indicate apoptosis. The sites of hemorrhagic necrosis are indicated by arrows.