Abstract
Macrophages in tumor microenvironment are mostly M2-polarized - and have been reported to promote tumorigenesis, which are also defined as tumor-associated macrophages (TAMs). Here, we examined the regulatory effects of Huaier extract on TAMs using RAW264.7 murine macrophage cell line. Our data demonstrated that Huaier extract could inhibit the infiltration of macrophages into tumor microenvironment in a dose-dependent manner. By performing RT-PCR, immunofluorescence and phagocytosis assay, we were able to find that Huaier extract could regulate the polarization of macrophages, with decreased M2-polarization and increased phagocytosis of RAW264.7 cells. Moreover, we identified that Huaier extract could suppress macrophages-induced angiogenesis by using HUVEC migration assay, tube formation and chorioallantoic membrane assay. Additionally, western blotting showed decreased expression of MMP2, MMP9 and VEGF with the use of Huaier extract. Finally, we found that Huaier extract could inhibit M2-macrophages infiltration and angiogenesis through treating 4T1 tumor bearing mice with Huaier extract. Our study revealed a novel mechanism of the anti-tumor effect of Huaier extract which inhibited angiogenesis by targeting TAMs. These findings provided that Huaier was a promising drug for clinical treatment of breast cancer.
Highlights
MethodsDulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco-BRL (Rockville, IN, USA)
Effects[9,10,11]
To test whether Huaier extract could inhibit tumor progression via the inhibition of M2 macrophages infiltration in tumor microenvironment, we first tested the influence of Huaier extract on macrophage motility
Summary
Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco-BRL (Rockville, IN, USA). Fetal bovine serum (FBS) was supplied by Haoyang Biological Manufacturer Co., Ltd (Tianjin, China). Anti-MMP2, anti-MMP9 antibodies were bought from Cell Signaling Technology (Beverly, MA, USA). AntiVEGF, anti-CD206 antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-CD31 antibody was provided by Immunoway Biotechnology Company (Newark, DE, USA). Anti-mouse IgG horseradish peroxidase (HRP) antibody was from ZhongShan Goldenbridge (Beijing, China). Fluorescein isothiocyanate–dextran and LPS were purchased from Sigma–Aldrich (St Louis, MO, USA). Murine INF-γ , IL-4 and IL-13 were bought from Peprotech (Rocky Hill, NJ, USA). The study was approved by the institutional guidelines of the Animal Care and Use Committee at Shandong University. All the methods used in our study were carried out in accordance with the approved guidelines
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