Ganoderma tsugae extract inhibits expression of epidermal growth factor receptor and angiogenesis in human epidermoid carcinoma cells: In vitro and in vivo
Introduction
The epidermal growth factor receptor (EGFR) belongs to the ErbB receptor kinase family, which includes ErbB1 (EGFR), ErbB2 (HER2/neu), ErbB3 and ErbB4. EGFR is overexpressed in many epithelial cancers, where the overexpression of EGFR in these carcinomas correlates with poor prognosis and decreased survival. Signaling through EGFR activation is often linked with tumor progression and metastasis, including proliferation, inhibition of apoptosis, invasion, and angiogenesis [1]. Blockage of EGFR activation causes the downregulation of the production of angiogenesis-related factors, such as vascular endothelial growth factor (VEGF), which results in the inhibition of angiogenesis [2], [3], [4], [5]. Angiogenesis is the formation of new blood vessels from pre-existing ones and is essential for the growth and persistence of solid tumors and their metastases [6]. In many cancers, VEGF, a multifunctional cytokine, and its receptors play an important role in primary tumor growth, angiogenesis and the development of metastases [7]. Therefore, inhibiting the expression of EGFR in tumor growth and in angiogenesis becomes a good target for human cancer therapy.
Ganoderma (also known as Lingzhi or Reishi) has been one of the most popular chemopreventive mushrooms in East Asia for centuries [8], [9], [10]. Among many bioactive components identified from Ganoderma, polysaccharides and triterpenoids are the two major ingredients for the treatment of various diseases, including cancer [10]. For example, some of the polysaccharides isolated from Ganoderma tsugae by water have been demonstrated to have anti-tumor activity on mouse cancer cells in vitro and in vivo[11], [12]. Although the pharmacological applications of the water extracts of G. tsugae have been extensively documented, little is known regarding its methanol extract in which methanol is mainly used to extract the triterpenoids [13], [14]. We have previously demonstrated that G. tsugae methanol extract (GTME) inhibits colorectal cancer cell proliferation in vitro and in vivo[15]. And the molecular mechanism(s) underlying the inhibition of the proliferation and angiogenesis of epidermoid carcinoma cells, however, remain to be determined.
In this study, we examined the effect of GTME on angiogenesis-related to the EGFR-overexpressing human epidermoid carcinoma A-431 cells. We found that GTME inhibits both the proliferation of A-431 cells and the capillary-like tube formation of human endothelial cells. Thus, we hypothesized that GTME may suppress EGFR expression and alter the malignant phenotypes mediated by EGFR in epidermoid carcinoma cells. Here, our results reveal that GTME can inhibit EGFR and VEGF expression in EGFR-overexpressing cancer cells. Furthermore, the cellular pathway from EGFR to VEGF leading to the inhibition of angiogenesis by GTME is also investigated on A-431 cells in this study. Finally, an animal model is also established to study whether the oral administration of GTME can suppress tumor growth as well as EGFR- and VEGF-induced angiogenesis in mice. Taken together, we attempt to provide the molecular mechanism by which the GTME inhibits the angiogenesis-related suppression of EGFR-mediated neovascularization.
Section snippets
Cells and chemicals
The human epidermoid carcinoma A-431 cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). The A-431 cells were cultured in DMEM with high glucose medium (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum in the humidified atmosphere of an incubator with 5% CO2 at 37 °C. Paclitaxel (Taxol) was purchased from Bristol-Myers Squibb (Bristol-Myers Squibb, Wallingford, CT), and Adriamycin (Doxorubicine) from Pharmacia (Pharmacia & Apjohn S.P.A.
Cytotoxic effect of GTME on A-431 cells
To verify that GTME reduced the cell proliferation of EGFR-overexpressing cancer cells, we first examined the effects of treatment with different concentrations of GTME (0.3, 0.75, 1.5, 2.25 and 3.0 mg/ml) for various treatment durations (24, 48 and 72 h) on human epidermoid carcinoma A-431 cells. After treatment, survival was inversely correlated with extract concentration and treatment duration (Fig. 1A). Since studies showed that EGFR-targeting drugs potentiated the cytotoxic effects of
Discussion
The medicinal mushroom Ganoderma sp. has long been used as a folk medicine in the Oriental region for the prevention and treatment of various diseases, including cancer [10]. Ganoderma contains numerous bioactive natural components and the two major categories of those are polysaccharides and triterpenoids, both of which are potent inhibitors of in vitro and in vivo tumor growth [8], [9], [10]. Several reports demonstrated that the anti-tumor effect of polysaccharides, usually extracted from
Conflict of interest statement
None declared.
Acknowledgements
This work was supported by the Department of Health (DOH93-TD-F-113-030 & DOH94-TD-F-113-023), Taiwan, ROC, and the China Medical University (CMU95-296), Taichung, Taiwan, granted to M.C.K.
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