Original Research Article
The anti-cancer activity of Antrodia camphorata against human ovarian
carcinoma (SKOV-3) cells via modulation of HER-2/neu signaling pathway
Hsin-Ling Yanga, Kai-Yuan Linb, Ying-Chen Juana, K.J. Senthil Kumare, Tzong-Der
Wayc, Pei-Chun Shena, Ssu-Ching Chend, and You-Cheng Hseue,*
aInstitute of Nutrition, China Medical University, Taichung, Taiwan
bDepartment of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan cDepartment of Life Sciences, China Medical University, Taichung, Taiwan dDepartment of Life Sciences, National Central University, Chung Li, Taiwan
eDepartment of Cosmeceutics, College of Pharmacy, China Medical University, Taichung,
Taiwan
*Corresponding author. Tel.: +886 4 22053366x5308; fax: +886 4 2207 808.
ABSTRACT
Ethnopharmacological relavence: Antrodia camphorata (AC) is well known in Taiwan as a
traditional Chinese medicinal fungus. However, the anticancer activity of AC against human HER-2/neu-overexpressing ovarian cancers is poorly understood.
Materials and methods: The aim of this study is to investigate whether a submerged
fermentation culture of AC can inhibit human ovarian carcinoma cell (SKOV-3) proliferation by suppressing the HER-2/neu signaling pathway. Cell viability, colony formation, DCFH-DA fluorescence microscopy, western blotting, HER-2/neu immunofluorescence imaging, flow cytometry, and TUNEL assays were carried out to determine the anti-cancer effects of AC.
Results: MTT and colony formation assays showed that AC induced a dose-dependent
reduction in SKOV-3 cell growth. Immunoblot analysis demonstrated that HER-2/neu activity and tyrosine phosphorylation were significantly inhibited by AC. Furthermore, AC treatment significantly inhibited the activation of PI3K/Akt and their downstream effector β-catenin. We also observed that AC caused G2/M arrest mediated by down-regulation of cyclin D1, cyclin A,
cyclin B1, and Cdk1 and increased p27 expression. Notably, AC induced apoptosis, which was associated with DNA fragmentation, cytochrome c release, caspase-9/-3 activation, PARP degradation, and Bcl-2/Bax dysregulation. An increase in intracellular reactive oxygen species (ROS) was observed in AC-treated cells, whereas the antioxidant N-acetylcysteine (NAC) prevented AC-induced cell death, HER-2/neu depletion, PI3K/Akt inactivation, and Bcl-2/Bax dysregulation, indicating that AC-induced cell death was mediated by ROS generation.
Conclusions: These results suggest that AC may exert anti-tumor activity against human
ovarian carcinoma by suppressing HER-2/neu signaling pathways.
HER-2/neu Ovarian cancer ROS
G2/M arrest
1. Introduction
Ovarian cancer is the fifth most common cancer among women worldwide and is the leading cause of death among gynecologic malignancies (Gari et al., 2006). Ovarian cancer predominantly affects elderly and middle-aged women, and its greatest incidences are reported
in North America and Northern Europe (Makar, 2000). However, ovarian cancer has a low
incidence in Asia, Africa, and Latin America (Stewart, 2012). Ovarian Cancer (OC) incidence rates from 1984-2006 is due to development of better diagnostic techniques and equipment (Chiang et al., 2010). The incidence of ovarian cancer increases with age; it is relatively rare in women younger than 30 years, although with increased modernization and urbanization, the rates appear to be increasing (Makar, 2000). Although scientific advancements have increased ovarian cancer survival rates, much research is still needed. Approximately and 25-80% of ovarian cancers express estrogen receptor (ER). However, the expression pattern may vary with cell types (Kalli et al, 2004). The current treatment of ER-positive tumors primarily relies on surgery to remove gross tumors, followed by treatment with anti-cancer agents that target the hormone dependence of these tumors, including aromatase inhibitors and anti-estrogens such as tamoxifen (Ao et al., 2011). However, anti-hormonal therapy is rarely used to treat epithelial ovarian cancers, but it is more often used to treat ovarian stromal tumors (Berek et al., 2000). Many women with ovarian cancer undergo anti-hormonal therapy that terminate ovarian function, resulting uncomfortable signs and symptoms, such as hot flashes, joint and muscular pain, bone thinning, and osteoporosis (Hervik and Mjaland, 2012). Therefore, the treatment of epithelial ovarian cancer still required non-side effect hormonal or chemotherapeutic agents. Human epidermal growth factor receptor-2 (HER-2/neu) is one of the most widely studied putative biological prognostic factors in human epithelial ovarian cancers (Graeff et al., 2009).
HER-2/neu, a proto-oncoprotein, belongs to the epidermal growth factor (EGFR) family, which consists of four receptors: EGFR (1/ErbB1), 2 (ErbB2), 3 (ErbB3), and HER-4 (ErbB) (Jiang et al., 2012). Since HER-2/neu overexpression is limited to 20-30% of many human cancers, including ovarian, breast, lung and gastric cancers, conflicting epidemiological data may reflect differing proportion of HER-2/neu positive cancers in the various studies (Aigner et al., 2000; Howe et al., 2001). HER-2/neu expression is associated with increased metastatic potential and angiogenesis, suggesting that the enhanced tyrosine kinase activity of HER-2/neu may play a critical role in the initiation, progression, and outcome of human ovarian cancers (Hsieh et al., 2004). Therefore, targeting HER-2/neu has been the main focus of ovarian cancer treatment, and the inhibition of HER-2/neu has become an increasingly important therapeutic target for HER-2/neu-overexpressing ovarian cancers.
Antrodia camphorata (AC) is a native Taiwanese medicinal mushroom that is popularly known
as “Niu Cheng Zhi” in Taiwan and grows in the inner sap of the tree Cinnamomum kanehira Hay (Lauraceae) (Hseu et al., 2002). AC has been used in traditional Chinese medicine for the treatment of food poisoning, drug intoxication, diarrhea, abdominal pain, hypertension, skin irritation and cancer (Yang et al., 2012). This medicinal mushroom is starting to attract interest because it possesses a number of bioactive components, including triterpenoids, polysaccharides, maleic/succinic acid derivatives, benzenoids and benzoquinone derivatives (Ao et al, 2009; Hseu et al., 2002; Yang et al., 2012). There is increasing evidence that AC possesses an extensive range of biological activities, including antioxidant, anti-inflammatory, hepatoprotective, anti-metastasis, anti-hypertensive, anti-hyperlipidemic, immunomodulatory and anti-cancer properties (Ao et al, 2009; Hseu et al., 2002; Lee et al., 2012; Yang et al., 2012). AC has low toxicity and is a non-mutagenic mushroom that efficiently reduces the
tumorigenicity of various cancers in vitro and in vivo. However, the anticancer activity of AC against human HER-2/neu-overexpressing ovarian cancers is poorly understood.
In this study, we investigated the effectiveness of the fermented broth of AC harvested from submerged cultures against HER-2/neu-overexpressing human epithelial ovarian cancer (SKOV-3) cells. We demonstrated that AC induced growth inhibition, cell-cycle arrest, and apoptotic induction of HER-2/neu-overexpressing SKOV-3 cells through intracellular ROS generation, suppression of the HER-2/neu signaling cascade, and disruption of the PI3K/Akt signaling pathway.
2. Materials and Methods 2.1. Reagents
Dulbecco’s Modified Eagle’s medium (DMEM), nutrient mixture F-12, fetal bovine serum (FBS), glutamine and penicillin/streptomycin were obtained from GIBCO BRL (Grand Island, NY). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC), p-iodonitrotetrazolium violet, and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Antibodies against phospho-tyrosine, p27, Cdk1, Cdk2, cytochrome c, Bcl-2, Bax, PARP, cyclin A, cyclin B1, Cdc25C, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Antibodies against HER-2/neu, phospho-PI3K, PI3K, phospho-Akt, Akt, β-catenin, caspase-9, caspase-3, cyclin D1, Cdk4, phospho-p38MAPK, p38MAPK, phospho-ERK, ERK, phospho-JNK, and JNK were obtained from Cell Signaling Technology, Inc. (Danvers, MA). Anti-phospho-HER-2/neu (Tyr1248) antibody was purchased from Millipore Corporation, Billerica, MA. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Calbiochem (La Jolla, CA). All
other chemicals were reagent grade or HPLC grade and were supplied by either Merck & Co., Inc. (Darmstadt, Germany) or by Sigma-Aldrich.
2.2. Preparation of the fermented culture broth of AC from submerged cultures
The AC culture was inoculated onto potato dextrose agar and incubated at 30 °C for 15–20 days. The whole colony was subsequently added to a flask containing 50 mL sterile water. After homogenization, the fragmented mycelial suspension was used as an inoculum. The seed culture was prepared in a 20-L fermenter (BioTop Process & Eqipment, Taiwan) agitated at 150 rpm with an aeration rate of 0.2 vvm at 30 °C. A five-day culture of 15 L mycelium inoculum was inoculated into a 250 L agitated fermenter (BioTop). The fermentation conditions were the same as those used for the seed fermentation, but the aeration rate was 0.075 vvm. The fermentation product was harvested at hour 331 and poured through a non-woven fabric on a 20-mesh sieve to separate the deep-red fermented culture broth and the mycelia; the culture broth was then centrifuged at 3000 g for 10 min followed by passage through a 0.22-m filter. The culture broth was concentrated under vacuum and freeze-dried to a powder. The yield of dry matter from the culture broth was 18.4 g/L. The experiments
were performed with 2–4 different batches of AC fermented culture (Hseu et al.,
2010). To prepare the stock solution, the powder samples were solubilized with DMEM containing 1% FBS (pH 7.4). The stock solution (1.6 mg/mL) was stored at -20 °C before its anticancer properties were evaluated. We refer to the fermented culture broth of Antrodia
camphorata as AC throughout the manuscript.
HER-2/neu-overexpressing human ovarian cancer cells (SKOV-3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and human ovarian surface epithelial (IOSE) cells were kindly provided by Dr Michael Chan, National Chung Cheng University, Taiwan, and cultured in DMEM/F12 supplemented with 10% heat-inactivated FBS, 2 mM glutamine and 1% penicillin-streptomycin-neomycin at 37 °C in a humidified incubator with 5% CO2. Cultures were harvested and monitored for changes in cell
number by counting cell suspensions using a hemocytometer with a phase contrast microscope. Cells were treated with 40-240 µg/mL of AC for 30 min-72 h, the incubation time varied depending on the assay. In additional experiments, cells were pretreated with 2.5 mM of NAC for 1 h followed by incubation with or without the indicated concentration of AC.
2.4. Cell viability assay
Cell viability was monitored by the colorimetric MTT assay. Briefly, SKOV-3 or IOSE cells (2.5 × 105 or 1 × 105 cells/well in a 24-well plate) were treated with AC (40–240 g/mL) for 24
h. Then, 0.5 mg/mL MTT in phosphate-buffered saline (PBS, 400 μL) was added to each well and incubated at 37 °C for 4 h. The MTT-generated violet formazan crystals were dissolved in 10% SDS (400 µL/well), and the absorbance was measured at 570 nm (A570). Cell viability was
calculated as (A570 of treated cells/A570 of untreated cells) × 100%.
2.5. Colony formation assay
Anchorage-independent growth was determined by colony formation in soft agar (Koleske et al., 1995). The assay was performed in 6-well plates (1 × 104 cells/well) with a
glutamine, 100 units penicillin and 100 g/mL streptomycin. This layer was overlaid with a second layer of 1 mL 0.35% agar (in DMEM containing 10% FBS, 1 mM glutamine, 100 units of penicillin and 100 g of streptomycin)
with a suspension of 1 × 104 cells/well. Fresh medium with AC (40–240 g/mL) was
then added to the plates every 72 h. The plates were incubated at 37 °C for 3 weeks, and the tumor colonies were analyzed with a microscope. Colonies with a diameter greater than 200 m were counted.
2.6. Measurement of ROS generation
Intracellular ROS generation was detected by fluorescence microscopy with 2′,7′-dihydrofluorescein-diacetate (DCFH-DA) as described previously (Lee et al., 2012). Briefly, SKOV-3 cells at a density of 1 × 105 cells/12 wells were cultured in DMEM/F-12
supplemented with 10% FBS. To evaluate the generation of ROS over time, cells were treated with 160 μg/mL of AC for 0, 1, 5, 10 or 15 min. The cells were then incubated with 10 μM DCFH-DA in culture medium at 37 °C for 30 min. The acetate groups on DCFH-DA were removed by an intracellular esterase, trapping the probe inside the cells. After loading, the cells were washed with warm PBS buffer. The production of ROS was measured by the change in fluorescence due to the intracellular accumulation of dichlorofluorescein (DCF) caused by oxidation of DCFH. Intracellular ROS, as indicated by DCF fluorescence, was measured by fluorescence microscopy (Olympus 1 × 71 at 200× magnification).
2.7. Fluorescence imaging of HER-2/neu
supplemented with 10% FBS in glass eight-well Tek chambers (Nunc, Denmark). After AC treatment for 24 h, the culture medium was removed, and the cells were washed with PBS, fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, washed, blocked with 10% FBS in PBS, and incubated for 2 h with an anti-HER-2/neu primary antibody in 1.5% FBS. The cells were subsequently incubated with a FITC-conjugated secondary antibody for 1 h in 6% bovine serum albumin (BSA) followed by nucleus staining with 1 μg/mL DAPI for 5 min. The stained cells were washed with PBS and visualized using a fluorescence microscope at 400× magnification.
2.8. Cell-cycle analysis
Cellular DNA content was determined by flow cytometric analysis with propidium iodide (PI)-labeled cells as previously described (Hseu et al., 2012). Cells were seeded at a density of 1.5 × 106 cells/10 cm dish and incubated overnight. The synchronization of the cell cycle was
achieved using a double thymidine block. Briefly, cells were treated with 3 mM thymidine in DMEM containing 10% FBS for 16 h. After treatment, the cells were washed twice with PBS and cultured in fresh medium for another 10 h. The cells were then blocked with DMEM containing 3 mM thymidine for 16 h. Cell-cycle-synchronized cells were washed with PBS and re-stimulated to enter the G1 phase together by addition of fresh DMEM containing AC (160
g/mL). The cells were harvested at 6, 12, or 18 h by trypsinization and then fixed in 3 mL of ice-cold 70% ethanol at -20 °C overnight. Cell pellets were collected by centrifugation, re-suspended in 500 μL of PI staining buffer (1% Triton X-100, 0.5 mg/mL RNase A and 4 μg/mL PI in PBS) and incubated at room temperature for 30 min. The cell-cycle progression was detected on a FACScan cytometer (BD Biosciences, San Jose, CA) equipped with a single
argon ion laser (488 nm). The forward and right-angle light-scattering, which correlate with cell size and cytoplasmic complexity, respectively, were used to establish size gates and exclude cellular debris from the analysis. The DNA content of 1 × 104 cells/analysis was
monitored using the BD FACSCalibur system. The cell-cycle profiles were analyzed with ModFit software (Verity Software House, Topsham, ME).
2.9. Quantification of apoptosis
Apoptotic cell death was measured using terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) with a fragmented DNA detection kit (Roche,
Mannheim, Germany). SKOV-3 cells (2 × 104 cells/well) were seeded in DMEM/F-12 medium
with 10% FBS in glass eight-well Tek chambers and treated with various concentrations of AC (40–240 μg/mL) for 24 h. After AC treatment, cells were washed with PBS twice, fixed in 2% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. The cells were then incubated with TUNEL reaction buffer in a 37 °C humidified chamber for 1 h in the dark, rinsed twice with PBS and incubated with DAPI (1 mg/mL) at 37 °C for 5 min; stained cells were visualized by fluorescence microscopy (200 × magnification).
2.10. Western blot analysis
SKOV-3 cells at a density of 1.5 × 106 cells/10 cm dish were incubated with various
concentrations of AC (40-240 µg/mL) for 24 h. After incubation, the cells were washed once in PBS, detached, pooled and centrifuged at 1500 × g for 5 min. The cell pellets were subsequently suspended in 100 μL lysis buffer containing 10 mM Tris–HCl, pH 8.0, 320 mM sucrose, 1% Triton X-100, 5 mM EDTA, 2 mM dithiothreitol and 1 mM phenylmethylsulfonyl
fluoride. The suspensions were sonicated and kept on ice for 20 min, then centrifuged at 15000 × g for 30 min at 4 °C. Total protein content was determined by Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) using BSA as a standard. Protein extracts were reconstituted in sample buffer (62 mM Tris–HCl, 2% SDS, 10% glycerol, 5% β-mercaptoethanol), and the mixture was boiled at 97 °C for 5 min. Equal amounts (50 μg) of denatured protein samples were loaded into each lane, separated by SDS-PAGE in an 8–15% polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes overnight. The membranes were blocked with 5% non-fat dried milk in PBS containing 1% Tween-20 for 1 h at room temperature, subsequently incubated with primary antibodies overnight, and further incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies for 2 h. Blots were visualized on an ImageQuant™ LAS 4000 mini (Fujifilm) system with SuperSignal West Pico chemiluminescence substrate (Thermo Scientific, IL).
2.11. Statistical analysis
The results are presented as the mean ± standard deviation (mean ± SD). All data were analyzed using analysis of variance followed by Dunnett’s test for pair-wise comparison. An asterisk indicates that the experimental values were significantly different from those of the control (*P < 0.05).
3. Results
3.1. AC treatment inhibits proliferation and tumorigenic ability of ovarian cancer SKOV-3 cells
treated with various concentrations of AC (40-240 µg/mL) for 24 h. MTT assay detected a significant reduction in cell viability after exposure to AC for 24 h. The cell viability was reduced into 97%, 83%, 58%, and 42% by 40, 80, 160, and 240 µg/mL, respectively, as determined by MTT assay, with an IC50 value of 196 µg/mL (Fig. 1A). In addition, the normal
human ovarian surface epithelial (IOSE) cells were exposed to AC (40-240 µg/mL) for 24 h, showed more then 80% of cell survival (Fig. 1B). These results confirm that AC treatment targets only the malignant calls. The level of colony formation ability of untreated ovarian cancer cells was directly proportional to the aggressive potential of the specific cell line (Nair et al., 2004). We next evaluated the ability of SKOV-3 cells to form colonies on 6-well culture plates in the presence or absence of AC for 3 weeks and observed their growth in soft agar. The aggressive human ovarian cancer SKOV-3 cells produced many colonies (Fig. 1C), whereas colony numbers were significantly (P<0.05) suppressed to 82 ± 7%, 50 ± 4%, 27 ± 6%, and 7 ± 1% by 40, 80, 160, and 240 µg/mL, respectively (Fig. 1C). At the highest concentration of AC (240 µg/mL), colony formation was reduced by over 90% compared to the untreated control cells (Fig. 1C). Reductions in colony number were accompanied by reductions in colony size in SKOV-3 cells. These data suggest that treatment of HER-2/neu-overexpressing ovarian cancer cells with AC decreases their rate of proliferation and tumorigenic ability.
3.2. AC-induced cell death is mediated by intracellular ROS generation in SKOV-3 cells
We have previously reported that AC treatment induces ROS generation in human breast cancer cells (MCF-7, MDA-MB-453, and BT474), which is proposed to be one of the early events in the activation of apoptotic signaling (Lee et al., 2012; Yang et al., 2006). In this study, we also examined the involvement of AC in ROS generation in SKOV-3 cells.
Fluorescence microscopy with DCFH-DA as a fluorescent probe was performed to estimate the intracellular ROS accumulation in SKOV-3 cells. Incubation of cells with AC (160 μg/mL for 0, 1, 5, 10, or 15 min) caused a significant increase in fluorescence, and the maximum level of ROS accumulation (p<0.05) was observed at 5 min after AC treatment (Fig. 2A). To further confirm that AC treatment induced ROS generation, SKOV-3 cells were pre-incubated with or without NAC (2.5 mM), a scavenger of ROS for 1 h and then treated with AC (160 g/mL) for 5 min. As shown in Fig. 2B, exposure of SKOV-3 cells to AC (160 µg/mL) led to a 5.3-fold increase in the ROS generation compared with control cells, whereas NAC pretreatment significantly (p<0.05) inhibited the increase in ROS generation, reducing ROS to the control level (Fig. 2B). To further demonstrate that AC-induced cell death is mediated by ROS generation, SKOV-3 cells were preincubated with NAC for 1 h and treated with or without AC (160 µg/mL) for 24 h. Cell viability was measured using MTT assay. As expected, NAC pretreatment significantly (p<0.05) prevented AC-induced cell death in SKOV-3 cells (Fig. 2C), which was concomitant with the inhibition of AC-induced ROS generation in SKOV-3 cells.
3.3. AC treatment modulates HER-2/neu protein expression by inhibiting its tyrosine phosphorylation
Activation of the HER-2/neu leads to autophosphorylation of the C-terminal tyrosines of the receptor and the recruitment to these sites of cytoplasmic signal transducers that regulate cellular processes, such as proliferation, inhibition of apoptosis and transformation (Olayioye, 2001). We first examined whether AC treatment altered HER-2/neu tyrosine kinase activity in SKOV-3 cells. The active/inactive status of the main autophosphorylation site of HER-2/neu
(Tyr1248) was semi-quantified by Western blot analysis using a specific anti-phospho HER-2/neu (Tyr1248) antibody. As shown in Fig. 3A, basal tyrosine kinase activity was observed in untreated control cells, whereas treatment of SKOV-3 cells with AC significantly decreased HER-2/neu tyrosine kinase activity in a dose- and time-dependent manner. We also observed that exposure of SKOV-3 cells to AC not only reduced HER-2/neu tyrosine kinase activity but further reduced the cellular content of HER-2/neu protein itself. The sustained reduction in HER-2/neu protein expression by AC was dose- and time-dependent (Fig. 3A). Taken together, these findings indicate that AC reduces the basal tyrosine kinase phosphorylation and the activation of HER-2/neu receptors in HER-2/neu-overexpressing ovarian cancer cells. To further confirm the reduction in HER-2/neu protein level by AC treatment, the immunofluorescence images of HER-2/neu expression were examined. Representative images of untreated SKOV-3 cells compared with cells treated with AC are shown in Fig. 3B. AC-treated cells exhibited lower levels of immunofluorescence at the plasma membrane, and fluorescence was replaced by diffuse cytoplasmic punctate staining. AC caused a significant reduction (p<0.05) and localization of membrane-bound HER-2/neu in SKOV-3 cells in a dose-dependent manner (Fig. 3B).
To further delineate the link between AC-induced ROS generation and HER-2/neu activation, SKOV-3 cells were preincubated with NAC for 1 h and treated with or without AC (160 µg/mL) for 24 h. The levels of tyrosine kinase phosphorylation and total HER-2/neu expression were measured by Western blot analysis. As shown in Fig. 3C, AC treatment caused a significant decrease in tyrosine phosphorylation and HER-2/neu expression to fold and 0.2-fold, respectively. However, pretreatment of SKOV-3 cells with NAC resulted in significant protection against the AC-induced reductions of tyrosine phosphorylation and HER-2/neu
expression (Fig. 3C).
3.4. AC treatment inhibits the activation of PI3K/Akt in SKOV-3 cells
HER-2/neu regulates tumor cell growth, and HER-2/neu overexpression renders ovarian cancer cells chemo-resistant. This effect is mediated by the PI3K/Akt signaling pathway, and human ovarian cancer cells with overexpression and amplification of HER-2/neu make increased use of the PI3K/Akt signaling pathway (Amler et al., 2012; Chuang et al., 2011). We therefore sought to determine the involvement of HER-2/neu in the activation of the PI3K/Akt signaling pathway in SKOV-3 cells. Large amounts of PI3K and Akt phosphorylation were observed in control cells, whereas AC treatment significantly inhibited their phosphorylation in HER-2/neu-overexpressing SKOV-3 ovarian cancer cells in a dose-dependent manner (Fig. 4A). The levels of total PI3K and Akt were unaffected by AC treatment. These data demonstrate that AC-induced HER-2/neu depletion and growth inhibition may be mediated by the down-regulation of PI3K/Akt signaling in HER-2/neu-overexpressing ovarian cancer cells.
Next we sought to confirm whether AC-induced ROS generation has any functional role in PI3K/Akt activation or inhibition. SKOV-3 cells were pretreated with NAC for 1 h and treated with or without AC (160 µg/mL) for 24. The phosphorylation of PI3K and Akt was monitored by Western blot analysis. AC treatment significantly inhibited the phosphorylation of PI3K and Akt, whereas the NAC pretreatment significantly protected the SKOV-3 cells against AC-induced inhibition of PI3K/Akt phosphorylation (Fig. 4B). In addition, the levels of total PI3K and Akt were unaffected by neither NAC nor AC. These data strongly suggest that AC-induced cell death is mediated by ROS generation.
3.5. AC treatment down-regulates the activation of β-catenin in SKOV-3 cells
Activated PI3K/Akt are critically involved in cell-cycle progression by phosphorylating and inactivating GSK-3β, thereby stabilizing nuclear translocation of β-catenin and increasing cyclin D1 transcription in human ovarian cancer cells (Rask et al., 2003). Therefore, to investigate the effect of AC on Wnt/β-catenin activity in human ovarian cancer cells, we treated SKOV-3 cells with AC for 24 h and measured the protein expression of β-catenin using cytoplasmic and nuclear extracts. As shown in Fig. 4C, strong β-catenin protein expression was observed in untreated cells. However, AC treatment caused a sustained decrease in β-catenin expression in both cytoplasmic and nuclear regions. Whereas, the loading controls β-catenin and histone levels were unaffected by AC. These data indicate that AC can inhibit Wnt/β-catenin signaling in human ovarian cancer cells.
3.6. AC treatment activates MAPK signaling pathways in SKOV-3 cells
MAP kinase family proteins, including p38, ERK, and JNK, play critical roles in cell fate. The p38 MAPK, ERK, and JNK modules are activated in response to cellular stress and seem to be exert both protective and pro-apoptotic functions (Boldt et al., 2002; Kim et al., 2008). To further examine whether AC treatment induces or inhibits the MAPK signaling pathways in human ovarian cancer cells, SKOV-3 cells were treated with AC (40–240 μg/mL) for 24 h, and the phosphorylation of p38, ERK, and JNK was assessed by Western blot analysis. As shown in
Fig. 4D, AC treatment significantly induced the phosphorylation of p38 MAPK, ERK, and JNK in a dose-dependent manner, whereas total p38 MAPK, ERK, and JNK remained unaltered by AC treatment. These results suggest that AC treatment in SKOV-3 cells up-regulates p38 MAPK, ERK, and JNK protein activities, which may act as pro-apoptotic inducers.
3.7. AC treatment induces G2/M cell-cycle arrest in SKOV-3 cells
We hypothesized that the reduction of cell viability in ovarian cancer cells by AC is due to cycle arrest. To test this hypothesis, we determined the effect of AC on the distribution of cell-cycle phases. As shown in Fig. 5A, exposure of SKOV-3 cells to AC resulted in a dose-dependent, progressive and sustained accumulation of cells in the G2/M phase. Furthermore, in
response to AC treatment, the percentage of cells in the G2/M phase gradually increased from
5% to 21% in SKOV-3 cells, whereas the percentage of those in the G1 phase was significantly
decreased (Fig. 5A).
To further examine the molecular mechanism(s) and underlying changes in cell-cycle patterns caused by AC treatment, we investigated the effects of AC on various cyclins and Cdks involved in cell-cycle regulation in SKOV-3 cells. AC treatment (40–240 g/mL) for 24 h caused a dose-dependent reduction of cyclin D1 and cyclin A/B1 expression in HER-2/neu-overexpressing SKOV-3 cells (Fig. 5B). Cyclin D1 serves as the regulatory subunit of Cdk4 and contributes to its stability. Therefore, next we assessed the effects of AC on Cdk expression. Treatment of SKOV-3 cells with AC resulted in a dose-dependent decrease in Cdk1 expression (Fig. 5B). Nevertheless, there was no change in the Cdk4, Cdk2, and Cdc25C protein levels (Fig. 5B). These results imply that AC inhibits cell-cycle progression by reducing the levels of cyclin D1, cyclin A/B1, and Cdk1 in SKOV-3 cells. In addition, PI3K/Akt signaling may contribute to the induction of cell-cycle progression by regulating the Cdk inhibitor p27. Therefore, we examined whether AC treatment induced p27 expression in 3 cells. As expected, a significant increase in p27 protein level was observed in SKOV-3 cells after exposure to AC (Fig. 5B). Thus, it can be speculated that AC treatment induces
G2/M cell-cycle arrest by increasing the Cdk inhibitor p27 in SKOV-3 cells.
3.8. AC treatment promotes apoptotic cell death in SKOV-3 cells
The PI3K/Akt cell survival pathway plays an important role in inhibiting apoptosis in HER-2/neu-overexpressing ovarian cancer cells (Lee et al., 2012), which prompted us to examine whether this pathway plays a role in induced apoptosis. We first assessed whether AC-induced cell death occurred through apoptotic induction. We used TUNEL staining to identify apoptotic cell death induced by AC. As shown in Fig. 6A, TUNEL-positive nuclei were found throughout the photomicrographs of the AC treatment group, whereas TUNEL-positive nuclei were undetectable in untreated control cells. Moreover, the number of TUNEL-positive nuclei significantly increased with AC concentration.
We further hypothesized that AC-induced apoptosis is mediated by mitochondrial pathways. Therefore, mitochondrial apoptosis was evaluated by directly measuring the release of mitochondrial cytochrome c into the cytosol by Western blot analysis. As shown in Fig. 6B, AC treatment caused a significant, dose-dependent increase in cytochrome c release from mitochondria, and the amount of cytochrome c in the cytoplasm was markedly increased, both of which show that AC caused mitochondrial membrane damage. Cytochrome c is involved in the activation of caspases that trigger apoptosis. Therefore, next we examined caspase-9 and caspase-3 cleavage by Western blot to evaluate caspase cascade activation in AC-induced apoptosis. Treatment of SKOV-3 cells with AC significantly induced the proteolytic cleavage of procaspase-9 and -3 into their active forms (Fig. 6B). In addition, PARP-specific proteolytic cleavage by caspase-3 is considered a biochemical characteristic of DNA damage. Our results demonstrate that AC treatment dose-dependently increased the cleavage of PARP (Fig. 6B).
Moreover, the regulation of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 plays a crucial role in cell homeostasis. Our results show that incubation of SKOV-3 cells with AC caused a dramatic reduction in the level of the anti-apoptotic protein Bcl-2 and increased the level of the pro-apoptotic Bax protein (Fig. 6B), which heterodimerizes with Bcl-2 to inhibit Bcl-2 activity. These results strongly indicate that AC-induced apoptosis is mediated by the dysregulation of Bcl-2/Bax expression. Therefore, we believe that the induction of apoptosis could be a major mechanism of AC-induced growth inhibition in SKOV-3 cells.
To determine whether there is a link between AC-induced ROS generation and Bcl-2/Bax dysregulation, SKOV-3 cells were preincubated with NAC for 1 h and treated with or without AC (160 µg/mL) for 24 h. The levels of Bcl-2 and Bax were measured by Western blot analysis. As shown in Fig. 6C, AC treatment caused a reduction in Bcl-2 protein and increased Bax protein. However, pretreatment of SKOV-3 cells with NAC resulted in significant protection against AC-induced up-regulation of Bax in SKOV-3 cells (Fig. 6C).
4. Discussion
Overexpression of HER-2/neu, a 185-kDa transmembrane kinase, is frequently observed in breast and ovarian cancer cells and implies a poor clinical diagnosis. HER-2/neu activates downstream signaling pathways, including the PI3K/Akt pathway, which mediates cell proliferation, survival and migration. The HER-2/neu oncogene encodes a receptor-like tyrosine kinase (p18) that has been extensively studied because of its role in several human carcinomas, including ovarian and breast carcinomas. In this study, AC-induced inhibition of cell proliferation, clonogenicity, and induction of apoptosis was observed in HER-2/neu-overexpressing human ovarian SKOV-3 cancer cells. We showed that AC treatment effectively
inhibited the growth of SKOV-3 cells, with an IC50 value of 196 µg/mL. We also demonstrated
that exposure of the HER-2/neu-overexpressing ovarian cancer cells to AC resulted in the induction of cell death mediated by ROS generation, HER-2/neu depletion, and down-regulation of PI3K/Akt signaling. Anti-HER-2/neu receptor therapy has been increasingly recognized as a potential treatment for HER-2/neu-overexpressing breast and ovarian cancer patients, as supported by recent advances in this direction. Several HER-2/neu tyrosine kinase inhibitors, including gefitinib and trastuzumab, are currently in pre-clinical and clinical development (Goyne and Cannon, 2012). However, phase II trials of gefitinib in refractory metastatic carcinomas yielded disappointing responses, and the course of treatment is expensive and requires multiple administrations of trastuzumab. The major observation reported in this study is that AC treatment effectively down-regulates HER-2/neu protein expression and tyrosine phosphorylation in HER-2/neu-overexpressing SKOV-3 cells. These data indicate that AC may be used as a chemo-preventive or chemotherapeutic agent against human ovarian cancers.
Naturally occurring phyto-compounds such as apigenin, rhein, sulforaphen, erucin, isothiocyanates, and anthocyanins, as well as several herbal products, potentially modulate the HER-2/neu signaling pathway in HER-2/neu-overexpressing human ovarian, breast, and bladder cancers in vitro and in vivo (Abbaoui et al., 2012; Chang et al., 2012; Hui et al., 2010; Mafuvadze et al., 2012; Shiu et al., 2009). In this study, we demonstrated that the fermented culture broth of AC exhibited significant growth inhibition by inhibiting HER-2/neu expression and tyrosine phosphorylation in HER-2/neu-overexpressing ovarian cancer cells. AC contains predominantly polysaccharides, triterpenoids, steroids, benzenoids, and maleic/succinic acid derivatives (Ao et al, 2009; Hseu et al., 2002; Yang et al., 2012). The reported yields of
polysaccharides, crude triterpenoids, and total polyphenols in the fermented AC broth are 23.2 mg/g, 47 mg/g, and 67 mg/g, respectively, whereas no polysaccharides, crude triterpenoids, or polyphenols have been detected in the dry matter of the culture medium (Hseu et al., 2002). It is reasonable to suggest, therefore, that AC metabolizes the culture medium and releases active components during fermentation. Further bioassay-directed fractionations to identify and purify the compounds responsible for the anti-ovarian-cancer effect of AC are warranted.
Activation by phosphorylation of the HER-2/neu receptor tyrosine kinase activates PI3K and Akt signaling, which induce cell growth and inhibit apoptosis (Amler et al., 2012; Chuang et al., 2011). In numerous tumor types, PI3K/Akt suppresses apoptosis and induces tumor cell survival by a variety of stimuli, including growth factor withdrawal and loss of cell adhesion (Datta et al., 1999). Overexpression of HER-2/neu activates the PI3K/Akt signaling pathway without exogenous ligand stimulation, and PI3K/Akt pathway activation also leads to delayed apoptosis (Zheng et al., 2004). In this study, AC treatment reduced the steady-state levels of total PI3K protein and phosphorylated Akt, indicating that the disruption of Akt activation plays a functional role in AC-induced apoptosis in HER-2/neu-overexpressing human ovarian cancer cells. The present study also suggests that AC-induced inhibition of cyclin A, cyclin B1, and cyclin D1 is directly proportional to the suppression of HER-2/neu and PI3K/Akt activation in human ovarian cancer cells. Taken together, these results suggest that HER-2/neu may regulate cellular cyclin A/B1/D1 via the PI3K/Akt pathway, implying that PI3K/Akt signaling predominantly contributes to cell-cycle progression in HER-2/neu-overexpressing ovarian cancer cells.
In the present study, we also demonstrated that AC treatment significantly inhibited β-catenin expression, which may contribute to its inhibitory effects on the Wnt/β-catenin pathway. Akt
phosphorylates several downstream substrates, including GSK-3β, and phosphorylated GSK-3β undergoes proteasomal degradation to allow β-catenin to translocate to the nucleus and co-activate the transcription of several oncogenes and cell-cycle regulatory genes, such as cyclin D1, c-Myc, and matrix metalloproteinases (MMPs). These target genes up-regulate tumor cell migration and decrease cell-cell adhesion (Benton et al., 2009). The two most important proteins for ovarian cancer seem to be HER-2/neu and cyclin D1. Both proteins have prognostic significance because they are frequently overexpressed and implicated in experimental models of ovarian cancer (Hashimoto et al., 2010; Meden and Kuhn, 1997). The interaction between HER-2/neu and cyclin D1 appears to have therapeutic potential because several naturally occurring phytochemicals or synthetic drugs can reduce cyclin D1 expression through the down-regulation of HER-2/neu signaling, and the anti-HER-2/neu monoclonal antibody trastuzumab (Herceptin®) reduces cyclin D1 protein levels in human ovarian cancer
cells (Abuharbeid et al., 2004; Gianolio et al., 2012; Menendez et al., 2005). Our results also demonstrate that AC treatment significantly inhibited SKOV-3 proliferation, which was associated with the inhibition of β-catenin activation and decreased expression of its transcriptional targets, including cyclin D1. Further studies are warranted to investigate the mechanisms underlying AC’s effects on β-catenin and the possible application of AC for ovarian cancer chemotherapy.
The critical role of MAPK family proteins in cell proliferation and apoptosis is evidenced by the observation that dysregulation of MAP kinase cascades can result in cell transformation and cancer (Dhanasekaran and Johnson, 2007). p38 MAPK has recently gained attention as a tumor suppressor that is activated upon cellular stress and often engages pathways that can block proliferation or promote apoptosis (Dayem et al., 2010). The activation of ERK primarily
involves a program of proliferation and survival, while the activation of JNK neither promotes nor inhibits proliferation or apoptosis. Transient activation of ERK and JNK leads to increased proliferation and survival of cancer cells, although inactivation of JNK in some instances may also promote tumorigenesis (Kennedy et al., 2007). Our results show that AC treatment increased the activation of p38 MAPK, ERK, and JNK. We believe p38 MAPK and JNK may be involved in AC-induced growth inhibition in SKOV-3 cells, whereas the role of ERK is unknown.
Eukaryotic cell-cycle progression is coordinated by the sequential activation of Cdks (cyclin-dependent kinases), the activation of which is (cyclin-dependent upon their association with cyclins (Johnson and Walker, 1999). Our study suggests that the marked reduction in cyclin A and B1 levels observed upon the inhibition of Cdk1 is involved in the transition from G2 to M phase.
Moreover, treatment of HER-2/neu-overexpressing ovarian cancer cells with AC down-regulated Cdk1 without altering Cdk2/4. Based on these results, we suggest that AC inhibits growth at the level of the G2-M phase transition. In contrast with our results, a previous report
showed that HER-2/neu-expressing human breast cancer MDA-MB-453 cells exposed to AC exhibited cell-cycle arrest at the G1-S transition through the down-regulation of cyclin D1, cyclin E1, and Cdk4, whereas Cdk1 and Cdk2 were unaffected (Boldt et al., 2002). These results indicate that the AC-induced cell-cycle arrest may vary from cell type to cell type, even among HER-2/neu-overexpressing cell lines. Cell-cycle progression is also coordinated by the balance between the cellular concentrations of Cdk inhibitors, including p27Kip1/Cip1 and p21WAF1
(Johnson and Walker, 1999). Loss of p27 protein activity is frequently observed in human cancers, including ovarian, prostate, gastric, colon, skin, lung and breast cancer, and is usually correlated with poor clinical outcome (Hsu et al., 2007). HER-2/neu-overexpression induces
down-regulation of p27Kip1 in a variety of cancer cell lines (Yang et al., 2000). The present
study shows that p27 protein expression was dose-dependently increased by AC. Thus, we suggest that the inhibition of cyclin A, B1, and D activity may be due to the increase of p27 expression.
Apoptosis-inducing agents are being investigated as tools for the management of cancer treatment. Activation of caspases, induction of apoptosis-inducing factors, chromatin condensation, and DNA fragmentation are the major biomarkers of cellular apoptosis (Lee et al., 2012). In the present study, TUNEL assays and Western blot analyses demonstrated that treatment of SKOV-3 cells with AC markedly induced apoptotic cell death associated with internucleosomal DNA fragmentation and caspase-3 and capase-9 activation. Caspase activation induced an elevation of cytochrome c in the cytosol, with a corresponding decrease in its mitochondrial level. These results show that the AC-induced increase in cytoplasmic cytochrome c was due to the release of mitochondrial cytochrome c into the cytoplasm. In mammalian cells, apoptotic cell death is critically governed by the balance between anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, and pro-anti-apoptotic proteins, including Bax and Bak (Coultas and Strasser, 2003). The induction of apoptosis by AC is associated with down-regulation of Bcl-2 and up-down-regulation of Bax expression in human breast cancer cell lines (Hseu et al., 2002; Lee et al., 2012). Similarly, the present study also shows that AC treatment down-regulates the expression of Bcl-2 and up-down-regulates Bax in SKOV-3 cells. These data indicate that AC treatment disturbs the Bcl-2/Bax ratio and thereby leads to apoptosis of HER-2/neu-overexpressing ovarian cancer cells.
Several anti-cancer drugs have been proposed to induce ROS generation by causing oxidative stress, which further leads to apoptosis in a variety of cancer cell lines (Ozben, 2007). Those
results are consistent with the hypothesis that AC-induced apoptotic cell death is driven by intracellular ROS generation because the anti-oxidant NAC prevents AC-induced cell death and Bcl-2/Bax dysregulation in SKOV-3 cells. By contrast, AC-induced ROS generation significantly inhibited HER-2/neu activity, as evidenced by inhibition of HER-2/neu tyrosine phosphorylation in SKOV-3 cells, whereas NAC treatment significantly prevented AC-induced HER-2/neu degradation and tyrosine phosphorylation, which strongly suggests that ROS could play a pivotal role in AC-induced growth inhibition in SKOV-3 cells.
In conclusion, AC-induced growth inhibition and apoptosis in SKOV-3 cells result from ROS generation, loss of HER-2/neu activation, and suppression of its downstream signaling, including the PI3K/Akt cascade. Our results also highlight the importance in ovarian cancer of HER-2/neu and PI3K/Akt signaling components, including β-catenin, cyclin D1, and p27KIP1,
which may serve as future targets for the development of therapeutic strategies. Moreover, this is the first report that demonstrated the effects of Antrodia camphorata on HER-2/neu-overexpressing human ovarian cancer cells. However, further in vivo studies are warranted to confirm the chemotherapeutic efficacy and safety of Antrodia camphorata.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Acknowledgements
This work was supported by grants NSC-99-2320-B-039-035-MY3, NSC-98-2320-B-039-037-MY3, CMU 98-C09, CMU 100-ASIA-13, and CMU 100-ASIA-14 from the National Science Council, China Medical University, and Asia University, Taiwan.
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Figure Legends
Fig. 1. AC inhibits cell proliferation and anchorage-independent growth of human ovarian
cancer (SKOV-3) cell lines. After incubation with various concentrations of AC (40–240 g/mL) for 24 h, cell viability of SKOV-3 (A) and Human ovarian surface epithelial (IOSE) cells (B) was examined by MTT assay. The number of viable cells after treatment is expressed as a percentage of the vehicle-only control, which was assigned as 100%. (C) Cells were assayed for their ability to proliferate and form colonies in soft agar. SKOV-3 cells were seeded onto 6-well plates in culture medium containing 0.35% low-melting agarose over a 0.7% agarose layer in the presence or absence of AC (40–240 g/mL) or vehicle control (PBS) and incubated for 3 weeks at 37 °C. The numbers of colonies >200 m in size were counted (at 40 × magnification). Colonies were subsequently stained with p-iodonitrotetrazolium violet (1 mg/mL), and colonies larger than 200 m were counted. The percentage of colony formation was calculated by defining the number of colonies in the absence of AC as 100%. The results are presented as the mean SD of three independent assays. *Significant difference in comparison to the control group (p<0.05).
Fig. 2. AC-induced ROS generation and its involvement in cell death. (A) SKOV-3 cells were treated with AC (160 g/mL) for 0, 1, 5, 10 or 15 min. The non-fluorescent, cell membrane-permeable probe DCFH-DA was added to the culture medium at a final concentration of 10 M for 30 min before the end of each experiment. DCFH-DA penetrated the cells, reacted with cellular ROS and was metabolized into fluorescent DCF, as indicated by DCF fluorescence, which was measured by fluorescence microscopy (200× magnification). The intracellular ROS level is expressed graphically as the relative fold increase over the control.(B) The antioxidant
N-acetylcysteine (NAC) prevents AC-induced cell death in SKOV-3 cells. Cells were
pretreated with 2.5 mM NAC for 1 h followed by treatment without or with AC (160 g/mL) and quantification of ROS after 5 min. (C) NAC prevents AC-induced cell death in SKOV-3 cells. Cells were pretreated with 2.5 mM NAC for 1 h followed by treatment without or with AC (160 g/mL) and quantification of cell viability after 24 h. The photomicrographs shown in this figure are from one representative experiment that was performed in triplicate. Each value is expressed as the mean ± SD (n=3). *Significant difference in comparison to the control group (p<0.05).
Fig. 3. Inhibitory effect of AC on phosphorylation of HER-2/neu (Tyr1248) and HER-2/neu expression in HER-2/neu-overexpressing human SKOV-3 cells. (A) Cells were incubated
without or with AC (40–240 g/mL) for 6-24 h. Immunoblotting was performed to measure total and tyrosine-phosphorylated HER-2/neu. Equal amounts of proteins (50 g) were resolved by 8–15% SDS-PAGE, with β-actin serving as a control. . Relative changes in protein bands were measured using densitometric analysis; the control was 1.0-fold, as shown immediately below the gel data. (B) Changes in the subcellular distribution of HER-2/neu after a 24-h exposure to AC. Cells were grown on coverslips and treated without or with AC (40–240 g/mL). Cells were fixed with 4% paraformaldehyde and stained with a HER-2/neu antibody followed by a fluorescein isothiocyanate-conjugated secondary antibody (green). The cells were photographed under fluorescence microscopy. (C) Cells were pretreated with 2.5 mM NAC for 1 h followed by treatment without or with AC (160 g/mL) and quantification of p-tyrosine and total HER-2/neu protein levels after 24 h.Relative changes in protein bands were measured using densitometric analysis; the control was 1.0-fold, as shown immediately below
the gel data. The results are presented as the mean SD of three independent experiments. *Significant difference in comparison to the control group (p<0.05).
Fig.4. (A) AC treatment suppressed the phosphorylation of PI3K/Akt in HER-2/neu-overexpressing SKOV-3 cells. Cells were treated with or without AC (40–240 g/mL) for 24 h. The levels of phosphorylated PI3K (p-PI3K) and Akt (p-Akt, pSer 473) were evaluated using PI3K and Akt phosphorylation-specific antibodies. The total PI3K and Akt levels were
assessed as controls. (B) SKOV-3 cells were pre-incubated with NAC (2.5 mM) and then
treated with or without AC (40–240 g/mL) for 24 h. The levels of phosphorylated and total PI3K and Akt were measured using Western blot analysis. (C) AC down-regulates β-catenin activity in SKOV-3 cells. Cells were incubated with control vehicle or AC (40–240 g/mL) for 24 h. Western blots show the effects of AC on the total protein contents of β-catenin in both
cytosol and the nucleus. (D) AC-induced activation of MAPK signaling pathways. SKOV-3
cells were treated with or without AC (40–240 g/mL) for 24 h. The levels of phosphorylated p38 (p-p38), ERK1/2 (p-ERK1/2), and JNK1/2 (p-JNK1/2) were evaluated using phosphorylation-specific antibodies. The total p38, ERK1/2, and JNK1/2 levels were assessed as controls. The photomicrographs shown in this figure are from one representative experiment that was performed in triplicate. Relative changes in protein bands were measured using densitometric analysis; the control was 1.0-fold, as shown immediately below the gel data.
Fig. 5. AC induces G2/M cell-cycle arrest in SKOV-3 cells. (A) Cells were treated with or
without 160 g/mL of AC for 6, 12 or 18 h, stained with PI and analyzed for cell-cycle phase by flow cytometry. Representative flow cytometry profiles are shown. (B) The effects of AC
(40–240 g/mL for 24 h) on cell-cycle regulatory protein levels were examined by immunoblotting. Equal amounts of protein (50 g) were resolved by 8–15% SDS-PAGE with β-actin as a loading control. Relative changes in protein bands were measured by densitometric analysis in which the control was 1.0-fold, as shown immediately below the gel data. The photomicrographs shown in this figure are from one representative experiment that was performed in triplicate.
Fig. 6. AC induces apoptosis in ovarian cancer cells. (A) SKOV-3 cells were exposed to AC (40–240 g/mL for 24 h), and the TUNEL assay was performed. The percentage of TUNEL positive cells were showed in the histogram. (B) Western blot analysis was performed to measure the protein levels of cytosolic and mitochondrial cytochrome c, procaspase-9 and -3,
PARP, Bax, and Bcl-2 in SKOV-3 cells after exposure to AC (40–240 g/mL for 24 h). (C)
Cells were pretreated with 2.5 mM NAC for 1 h followed by treatment without or with AC (160 g/mL) and quantification of Bax and Bcl-2 after 24 h using Western blot analysis. Relative changes in protein bands were measured by densitometric analysis in which the control was 1.0-fold, as shown immediately below the gel data. The photomicrographs shown here are from one representative experiment repeated two times.
