Elsevier

Phytomedicine

Volume 20, Issue 10, 15 July 2013, Pages 874-882
Phytomedicine

Elucidating the inhibitory mechanisms of the ethanolic extract of the fruiting body of the mushroom Antrodia cinnamomea on the proliferation and migration of murine leukemia WEHI-3 cells and their tumorigenicity in a BALB/c allograft tumor model

https://doi.org/10.1016/j.phymed.2013.03.008Get rights and content

Abstract

The aim of this study was to explore whether the ethanolic extract of Antrodia cinnamomea (EEAC), a medical mushroom form Taiwan, could affect the proliferation and migration of WEHI-3 cells in vitro and to explore the antitumor effects of EEAC in BALB/c mice engrafted with WEHI-3 cells. The results showed that EEAC inhibited the proliferation of WEHI-3 cells, resulting in the accumulation of cell in G0/G1 and G2/M phases, as determined by flow cytometry. Moreover, EEAC markedly reduced the migration of WEHI-3 cells, as determined by a transwell assay. Treatment of WEHI-3 cells with EEAC also decreased MMP-9 protein expression and enzyme activity. The protein levels of p-Akt, p-ERK1/2 were also decreased, whereas the expression of p21 and p27 was increased. Furthermore, in an in vivo model, EEAC treatment reduced the infiltration of WEHI-3 cells into the liver and spleens and decreased tumor growth. Other bioactive compounds, such as cordycepin and zhankuic acid A, have been demonstrated to reduce the expression of MMP-9, cyclin E, cyclin D1 and to increase the expression of p21, p27. This is the first study to investigate that the mechanisms by which EEAC reduce the proliferation and migration of WEHI-3 cells in vitro, as well as the ability of EEAC to reduced infiltration of WEHI-3 cells into the liver and spleen in vivo. The results suggest that EEAC may prove to be useful in future antileukemic therapies.

Introduction

The survival of patients with acute myeloid leukemia (AML) is poor, with only 21.4% and 18.7% of patients surviving for 5 and 10 years after diagnosis, respectively (Pulte et al. 2010). In clinical trials, arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) have been shown to be efficacious in the treatment of patients with both newly diagnosed and relapsing acute promyelocytic leukemia (APL), a subtype of AML, and standard AML (Amadori et al. 2005). However, in clinical trials, single agent treatment with ATO has not demonstrated any significant benefits in a variety of non-APL hematological malignancies (Lu et al. 2012b). Additionally long-term treatment with ATRA and ATO can result in several serious side effects, including hypertension (Li et al. 2010), interstitial pulmonary infiltrates, pleural and pericardial effusion, dyspnea, episodic hypotension and acute renal failure (Patatanian and Thompson 2008). Thus, new therapeutic agents are needed for the treatment of AML.

It is generally believed that the proliferation and migration of leukemia cells are involved in tumor growth and invasion. Therefore, modulation of the growth and migration properties of leukemia cells has important therapeutic implications. To explore this possible approach, we investigated the ability of Antrodia cinnamomea (AC), a medical mushroom from Taiwan, to modulate the proliferation and migration of leukemia cells in vitro, as well as the antitumor effects of AC in vivo.

Traditionally, AC has been used as a health food to prevent inflammation, hypertension, itchy skin, and liver cancer (Liaw 1985). There is increasing evidence that AC possesses an extensive range of biological properties, including protection against hepatotoxicity (Ao et al. 2009), anti-inflammatory properties (Chen et al. 2007), and antioxidant properties (Yang et al. 2006). Several studies have suggested that the extracts from the mycelia and the fruiting bodies of AC could be used as potential chemotherapeutic agents against various cancers, including hepatoma, prostate, colon, breast, bladder, and lung cancer (Hseu et al., 2008, Lin et al., 2010, Wu et al., 2006, Yang et al., 2011, Yeh et al., 2009). Our previous studies have identified that three main constituents of the EEAC, including adenosine, cordycepin and zhankuic acid A in a HPLC/Mass-fingerprint analysis (Chen et al. 2012b). However, the antimetastatic and antitumor effects of the fruiting bodies of AC in acute myeloid leukemia are still unclear. The present study was designed to explore the mechanisms of the antimetastatic effects of the ethanolic extract of AC (EEAC), cordycepin and zhankuic acid A on the WEHI-3 cell line, as well as the antitumor effects of EEAC in BALB/c mice injected with WEHI-3 cells.

Section snippets

Materials

Penicillin G, M199 (31100-035), FBS (10099-141) medium and streptomycin were obtained from Invitrogen (Carlsbad, CA, USA). Primary antibodies against phosphorylated-ERK1/2(p-ERK1/2, #sc-7383) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phosphorylated-Akt (p-Akt, #ab28821), MMP-9 (#ab58803), p21 (#ab7960) and β-actin (#ab8226) were purchased from Abcam (Cambridge, MA, USA). Antibodies against p27 (GTX100446), Cyclin D1 (GTX27958) and Cyclin E (GTX27959)

The effect of EEAC extract on the proliferation of WEHI-3 leukemic cells

The anti-proliferative effect of EEAC was analyzed using WEHI-3 cells, which were treated with 10% FBS to stimulate cell growth. The treatment of WEHI-3 cells with 6.25 to 25 μg/ml EEAE for 24 h significantly suppressed their proliferation, with a 50% inhibitory concentration (IC50) of 15 μg/ml (Fig. 1A). In contrast, HUVEC showed decreased proliferation only when incubated with EEAC at 40–60 μg/ml for 24 h, and HUVEC cell viability was not significantly affected by lower concentrations of EEAC,

Discussion

Abnormal cell proliferation and metastasis play critical biological roles in clinical cancer cases. Metastasis, the invasion of cancer cells into other organs, is a complicated process and is an important and characteristic step in cancer progression, resulting in poor clinical outcome. As such, a candidate drug that efficiently inhibits tumor growth and the invasion and migration of cancer cells may suppress cancer progression and metastasis, potentially even reducing mortality. In the present

Conflict of interest

All authors reviewed the manuscript, agreed with its contents, declared that they do not have any conflicts of interest in regard to the subject matter or materials in the research and approved its submission for publication.

Acknowledgements

The authors wish to thank Yu-Hsuan Lin, Tai-Yun Wu and Pei-Hua Tu for assistance with the animal experiments. This work was supported by grants from the China Medical University and the National Science Council (CMU98-NCTU-06, NSC97-2320-B-039-013-MY3).

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