The effect of the ethanol fraction of the mycelia of
Antrodia camphorata on human A549 cell
Ting-Feng Wu, Professor and Zhen-Lin Chang
Department of Biotechnology, Southern Taiwan University
Abstract
Antrtodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in
preventing diseases. In this study, in order for the fractionation, the mycelia were extracted with the order of hexane,
ethylacetate, ethanol and water respectively. Each fraction was tested for the inhibitory ability against human A549
cells and the expression of galectin-1 genes. The results of MTT assays and the western blotting with the galectin-1
antibody demonstrated that the ethanol fraction had the best the depressive effect on the proliferation of human
A549 cells and the expression of galectin-1 genes.
Keywords: Antrodia camphorata, ethanol fraction, galectin-1
Introduction
Basidiomycete Antrodia camphorata in Polyporaceae, a native fungus in Taiwan, is commonly used in
Taiwanese folk medicine. The fruit body of A. camphorata is rare and expensive because it grows only in the inner
heart wood wall of the endangered species Cinnamonum kanehirai and cultivation is extremely difficult.
Additionally, bio-active ingredients of its pharmacological functions, including triterpenoids, sesquiterpene lactone,
steroid and polysaccharide [2-5, 20], have been analyzed.
The influences of A. camphorata on a variety of biological functions have been studied. Peng et al. (2006)
demonstrated that A. camphorata crude extract (ACCE) can inhibit the proliferation of superficial and invasive
bladder transition cell carcinoma (TCC) cell lines and retard the migration of invasive T24 cells [11,12]. Song et al.
(2005) reported that the methanol extract of mycelia (MEM) from the submerged culture of A. camphorata can
induce the apoptosis of HepG2 cells possible via Fas pathway [14,17]. Hseu et al. (2004) indicated that the
fermented broth of A. camphorata can evoke the apoptosis of HL-60 and MCF-7 cells [6, 18]. The experimental
results with the fruit bodies of A. camphorata showed that the ethylacetate extracts has the apoptotic effect on the
HepG2 cells via increasing the Fas/APO-1 as well as membrane-bound Fas and initiating the mitochondrial
apoptotic pathway [9].
In addition to the anticancer activities, A. camphorata possess other pharmacological activities. Song et al.
(2002) evaluated the antioxidant and free radical scavenging activities of various preparations of mycelia and
found that the dry matter of fermented filtrates (DMF) has the strongest inhibition of lipid peroxidation and DMF
as well as water extract (WEM) has the noticeable free radical scavenging activities [15]. Anti-oxidation studies
showed that the aqueous extracts of mycelia can protect the erythrocyte from the damage induced by aqueous
preoxyl radical and it also can alleviate the depletion of cytosolic glutathione [7]. Song et al. (2003) reported that
the DMF can markedly reduce the H2O2-induced lipid peroxidation and protect the rat liver from the CCl4-induced
damage by up-regulating hepatic GSH-dependent enzymes [16]. The results of lipopolysaccharide-induced
Raw264.7 cells treated with culture broth of A. camphorata showed that the culture broth can lower prostaglandin
E2 and nitric oxide production and the expression of iNOS as well as cyclooxygenase-2, suggesting the culture
broth possess the anti-inflammatory activities [8].
Most of the studies investigating the pharmacological activities of A. camphorata hitherto utilize the crude
extracts. However, only few studies have harnessed the partially or pure compounds isolated from the crude
extracts to elucidate the therapeutic effects of A. camphorata. Shen et al. purified Zhankuic acid from the fruit
bodies to study its anti-inflammatory activities and observed that Zhankuic acid can decrease the fMLP or
PMA-induced ROS production in the peripheral human neutrophils and inhibit the firm adhesion of neutrophils
[13]. Five new maleic and succinic derivatives isolated from the mycelia by Nakamura et al. are found to show the
cytotoxicity against the LLC tumor cell lines [10]. Chen et al. purified three new dipterpenes together with four
known diterpenes from the fruit bodies and showed that five of these compounds have the neuroprotective abilities
[1].
In our previous studies [20], we found that the ethanol extracts of A. camphorata (SACE) can diminish the
expression of human galectin-1, human eukaryotic translation initiation factor 5A (eIF5A), human Rho GDP
dissociation inhibitor , human calcium-dependent protease (calpain) small (regulatory) subunit and human annexin
V genes in human A549cells. Among these, galectin-1 has been demonstrated to bind H-Ras to stimulate the
transformation [22] and be involved in the escape of tumor cell from the surveillance of the immune system [23].
Calpain has been demonstrated to play a role in the degradation of p53 [24]. Atencio et al. (2000) reported that
calpain inhibitor 1 can activate p53-dependent apoptosis in tumor cell lines [25]. Mackeigan et al. (2003) discovered
that in Taxol/MEK inhibitor-treated lung cancer H157 cell expression of Rho-GDP inhibitor  is diminished and
Rho-GDP inhibitor  expression knockdown by siRNA invokes the apoptosis of H157 cells [26]. A special amino
acid hypusine is necessary for the functionality of eIF-5A [27, 28]. Many works have indicated that downregulation or inhibition of hypusine synthesis impede cancer cell growth, including lung cancer cell and pancreatic
cancer cell [29-32], strongly indicating that the eIF-5A may be an appropriate new target for cancer intervention.
Taken together, it is reasonable to interpret that SACE induces the apoptosis of human A549cell via the downregulation of these tumor-related genes.
Because SACE is a crude extract, the components that affect the expressions of five tumor-related genes are not
known. Therefore, this study is intended to purify the fraction with the inhibitory effects against the A549 cells and
to the expression of galectin-1 gene.
Material and Methods
Fractionation of the mycelia
The mycelia will be extracted at the ratio of 1:5 (w/v) for 16 hours with shaking with the order of hexane,
ethylacetate, ethanol and water (Figure 1). After extraction, the mixture is filtered. The residual of each extraction is
extracted with the next solvent and the filtrate is dried using frozen-vacuum dryer. The powder is stored at -20C
until use.
Cell lines
Human non-small cell lung carcinoma A549 cell and primary human fetal lung fibroblast MRC-5 were purchased
from ATCC. Both A549 and MRC-5 cells were cultured at 37C in DMEM (Invitrogen Life Technologies Inc.,
Grand Island, NY), supplemented with 10% fetal bovine serum (Biological Industries Ltd., Kibbutz Beit Haemek,
Israel).
MTT assay
Various concentrations of each fraction as depicted in Fig. 2 were added to a 96-well plate already loaded with
3,000 A549 cells per well. After treatment for 72 hours, 20 l of MTT solution (Merck, Damstadt, German) (5
mg/ml PBS) was added to each well and the plate was incubated at 37ºC for 4 hours. After medium removal, 200 l
of DMSO was added to each well and the plate was gently shaken for 5 minutes. The absorbance was determined at
540 nm. Quadruplicate wells were applied to each concentration for 72 hours as shown in Fig. 2. The solvent
carrier-treated A549 cells were employed as the control.
Western blotting
After treatment of A549 cells with 1 mg/ml for 8 hours, the cells were harvested and lysed in the sample buffer
(0.1M Tris, pH 6.8, 2 % SDS, 0.2% -mercaptoethanol, 10% glycerol and 0.0016% bromophenol blue). Total cell
lysates (50 g of protein) were separated using 10% acrylamide gel electrophoresis and transferred onto the PVDF
membrane (Stratagene, La Jolla, CA). The membrane was blotted with primary antibody overnight followed by
incubation with HRP-conjugated secondary antibodies (1:15,000), and visualized using chemiluminescence
(Amersham-Pharmacia Biotech Inc., Piscataway, NJ). The antibodies were purchased from the following sources:
actin from Chemicon international and the human galectin-1 from CytoLab Ltd.Temecula, CA.
Results and discussion
In this study, the compounds present in the A549 cells were fractionated dependent upon the polarity. The mycelia
were extracted with the order of hexane, ethylacetate, ethanol and water respectively (Figure 1). Each fraction was
tested for the inhibitory effect against A549 cells. As shown in the Fig. 2 the ethanol fraction had the most inhibitory
effect on the A549 cells. This result was consistent with our previous results.
Since the ethanol fraction has the most depressive effect on A549 cells, this fraction was examined for the
inhibitory impact on the expression of galectin-1 gene. The results of western blotting with gaelctn-1 antibody
revealed that the ethanol fraction could diminish the expression of galectin-1 gene at 1mg/ml for 8-hour treatment
(Figure 3). Taken together, the results of this study implicate that the ethanol fraction contains some compound(s)
which can control the proliferation of A549 cells and diminish the expression of galectin-1 gene.
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